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This study aimed to promote the valorization of lupin seeds by extracting both non-polar and polar fractions to produce a protein-rich flour suitable for food applications. Green extraction methods such as Supercritical Fluid Extraction (SFE) and SFE followed by gas-expanded liquid extraction with ethanol/CO 2 mixtures were employed. SFE yielded lupin oil with extraction yields ranging from 2.27 ± 0.02 to 4.5 ± 0.2 %, significantly influenced by temperature (40 and 60 °C) and pressure (150-350 bar). SFE extracts exhibited higher tocopherol concentration, particularly α-tocopherol (116.7-296.9 µg/g oil) and γ-tocopherol (2006-4749 µg/g oil), compared to the Bligh and Dyer (B&D) method. The fatty acid profiles were similar, although they differed slightly in composition, with the extracts obtained by SFE having higher proportions of unsaturated fatty acids (UFA) and lower proportions of saturated fatty acids (SFA). Ethanol proportion positively correlated with extraction yield (r = 0.991), resulting in higher recovery of polar lipids (PL). However, increasing ethanol percentage decreased the phenolic compounds content and antioxidant activity assessed by DPPH radical scavenging method. SFE produced lupin flour with 36 % protein content, increased by 11 % post-extraction. Ethanolic extraction also increased protein concentration, albeit less pronounced (6.8-11 % increase post-sequential extraction). Essential amino acids consistently increased post-SFE, highlighting the potential of this sustainable method to yield protein-rich flour free of non-GRAS (Generally Recognized as Safe) solvents and containing compounds essential for human health. SDS-PAGE analysis showed consistent protein profiles across all extracted flours, while FTIR assessment revealed changes in the secondary structure of proteins induced by SFE and SFE followed by gas-expanded liquid extraction processes. These findings highlight the potential of this approach to enhance the nutritional and commercial value of lupin-based products while promoting sustainable food processing practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Multiple sclerosis (MS) is a neurodegenerative disease, with inflammation and oxidative stress in the central nervous system being the main triggers. There are many drugs that reduce the clinical signs of MS, but none of them cure the disease. Food proteins have been shown to contain encrypted peptides that can be released after hydrolysis and exert numerous biological activities. Recently, we have demonstrated the anti-inflammatory and antioxidant activities of a lupin protein hydrolysate (LPH) both in vitro and in vivo. Therefore, the aim of this study was to evaluate whether LPH is capable of reducing the clinical signs of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE was induced in female C57BL/6N mice and they were treated intragastrically with LPH (100 mg/kg) or vehicle (control group) from day 0 (prophylactic approach) or from the onset of the disease (day 12 post-induction; therapeutic approach) and the clinical score of each mouse was recorded daily. Prophylactic treatment with LPH reduced the clinical score of the mice compared to the control group, as well as the maximum and cumulative scores, without changing the day of onset of the symptoms while the therapeutic intervention did not significantly improve the severity of the disease. For the first time, we demonstrated that prophylactic administration of LPH reduces the severity of MS, suggesting a potential nutraceutical or new functional foods in neuroinflammation. However, further studies are needed to confirm this nutritional effect in a clinical context.

The COVID-19 pandemic has highlighted the urgent need for effective antiviral agents. One such promising drug is nirmatrelvir (NIRMA), which has demonstrated the ability to inhibit viral replication and reduce hospitalization and mortality rates. In the field of analytical chemistry, carbon quantum dots (CQDs) derived from fruit peel waste have gained attention for their eco-friendly and cost-effective properties. In this study, CQDs were synthesized from the peel biomass waste of yellow lupin and utilized for the spectrofluorometric determination of NIRMA in pharmaceutical tablets. The proposed method offers simplicity and speed compared to existing methods. The synthesis process involved washing and grinding the peel waste, followed by heating in propylene glycol and purification using a dialysis membrane. The resulting CQD solution showed fluorescence at an emission wavelength of 411 nm upon excitation at 325 nm. The validation of the method, including linearity, range, limit of detection and quantification, accuracy, precision, and robustness, was conducted following ICH guidelines. The fluorescence intensities exhibited a linear relationship with increasing NIRMA concentration in the range of 15-150 µg/mL. The limit of detection found to be 4.0 μg/mL, with %relative standard deviations ≤ 1.46. This spectrofluorometric method represents a novel approach for the determination of NIRMA using CQDs derived from yellow lupin peel waste., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Evidence-based therapies to manage the clinical signs of intoxication caused by toxic plants in livestock are lacking. For that reason, the aim of this work was to develop a drug-based intervention for the management of clinical signs of piperidine alkaloid intoxication in livestock. The actions of anabasine, coniine, γ-coniceine, and two total alkaloid extracts from Lupinus sulphureus were compared in the presence and absence of the nicotinic acetylcholine receptor partial agonist varenicline in RD cells, mice and goats. Pretreatment of RD cells with 10.0 μM varenicline significantly shifted the anabasine fifty percent effective concentration (EC 50 ) value to a greater concentration and blocked the response of the cells to coniine. γ-coniceine did not have any effect on RD cells as measured by membrane potential sensing dye. Swiss Webster mice median lethal dose (LD 50 ) values for anabasine, coniine, γ-coniceine were 1.5, 5.5, and 3.7 mg/kg respectively, and pretreatment with 10.0 mg/kg i. p. dosed varenicline shifted the LD 50 values to 4.2, 9.1, and 4.3 mg/kg respectively. The rodent LD 50 value of the Pendelton, WA L. sulphureus quinolizidine alkaloid extract was shifted to a lesser concentration by varenicline while the Ritzville, WA L. sulphureus piperidine alkaloid extract was shifted to a greater concentration by varenicline. The clinical signs of intoxication in goats orally dosed with Conium maculatum were exacerbated by 0.5, 1.0 and 10.0 mg/kg i. v. dosed varenicline. These results suggest that varenicline was effective at shifting piperidine alkaloid EC 50 values in RD cells and increasing piperidine but not quinolizidine alkaloid LD 50 values in mice and was not useful at managing the clinical signs of poison hemlock intoxication in goats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [Journal name] and was not involved in the editorial review or the decision to publish this article., (Published by Elsevier Ltd.)

Lupins are an excellent source of protein which can be used to obtain protein isolates with potential use in the food industry. Some studies use supercritical CO 2 (SC-CO 2 ) to defat legume flours, but no study analyzes the effect of applying this technology directly to the protein isolate. This article has proposed the use of SC-CO 2 to improve lupin protein isolate (LPI) quality. SC-CO 2 increased the LPI purity while reducing oil and other antitechnological factors (saponins and polyphenols). The treatment significantly improved the LPI color due to the elimination of the lipid fraction and lipophilic pigments (carotenoids). No changes in amino acid contents or chemical score were observed due to the SC-CO 2 . Finally, the treatment improved or did not affect the main LPI technofunctional properties. Therefore, SC-CO 2 is a promising technique to enhance the quality of protein isolates, without affecting or improving their functional properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lupin ( Lupinus angustifolius L.) proteins are potential sources of bioactive peptides (LBPs) that can inhibit dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE-I) activity. However, the capacity of different enzymes to release LBPs, the pharmacokinetic and bioactivities of the peptides released, and their binding affinities with the active sites of DPP-IV and ECA-I are topics scarcely addressed. Therefore, we used in silico hydrolysis (BIOPEP-UWM platform) with various enzymes to predict the release of LBPs. Among the bioactive peptides identified in lupin proteins (n = 4813), 2062 and 1558 had DPP-IV and ACE-I inhibitory activity, respectively. Ficin, bromelain, and papain released the highest proportion of ACE-I (n = 433, 411, and 379, respectively) and DPP-IV (n = 556, 544, and 596, respectively) inhibitory peptides. LBPs with favorable pharmacokinetics and gastrointestinal stability tightly interacted with the active sites of ACE-I (-5.6 to -8.9 kcal/mol) and DPP-IV (-5.4 to -7.6 kcal/mol). Papain generated the most bioavailable LBPs (n = 459) with ACE-I (n = 223) and DPP-IV (n = 412) inhibitory activity. These peptides were non-toxic and gastrointestinal digestion stable. Notably, papain-based hydrolysis released some LBPs (n = 270) that inhibited both ACE-I and DPP-IV. Plant protease-based hydrolysis is a promising approach for producing lupin hydrolysates with ACE-I and DPP-IV inhibitory activities.

The research aimed to assess the effects of incorporating germinated Lupinus angustifolius flour into corn extrudates for different periods (3, 5, and 7 days), focusing on starch digestibility, morphological structure, thermal, and pasting properties. Extrudate with germinated lupinus flour for 7 days (EG7) significantly increased the content of slowly digestible starch up to 10.56% (p < 0.05). Crystallinity increased up to 20% in extrudates with germinated flour compared to extrudates with ungerminated flour (EUG), observing changes at the molecular level by FTIR that impact the thermal and pasting properties. X-ray diffraction revealed angles of 2θ = 11.31, 16.60, 19.91, and 33.04 as a result of the germination and extrusion processes. Microstructural analysis indicated starch-protein interactions influencing changes in calorimetry, viscosity, X-ray diffraction, and digestibility. PCA allowed establishing that the addition of germinated flours significantly affected the properties and microstructural characteristics of extruded products, potentially affecting digestibility and nutritional quality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lupins, and other legumes, have attained international interest due to their reported remarkable health benefits. Currently, the seed coats are discarded as waste or animal feed. The research presented here summarizes the potential for incorporating the seed coats into 'whole grain' foods. We aimed to identify metabolites found in the seed coats of nine commercial Australian cultivars of lupin (Lupinus angustifolius and L. albus species), and to evaluate and compare their functional, nutritional, antioxidant, and antidiabetic properties, along with in silico exploration of mechanisms of action for selected identified secondary metabolites. The seed coats were found to contain 79 to 90% dietary fibers and substantial quantity of essential macrometals. LC-QTOF MS-based, untargeted bioactive metabolite profiling explored a total of 673 chemical entities, and identified 63 bioactive secondary metabolites including: biophenols, unsaturated fatty acids, triterpenoids, alkaloids, and dietary prebiotics (insoluble fibers). The seed coats from these nine cultivars show substantial antioxidant activity. The cultivars of L. angustifolius inhibit α-amylase and α-glucosidase significantly in vitro. Moreover, in silico docking and dynamic simulation along with ADME/T analysis suggest that quercetin 3-methyl ether and 8-C-methylquercetin 3-methyl ether as molecules, novel in lupin seed coats, are responsible for the α-amylase and α-glucosidase inhibition. The findings indicated that lupin seed coats might be beneficial food components, rather than be discarded as 'mill waste'., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In the current environment whereby new sources of proteins are extracted from plant material, it is also important to study the potential use of the resulting side streams. Although a number of studies have been conducted on various polysaccharides extracted from plant raw material, a polysaccharide fraction extracted from lupin bean is yet to be explored, in spite of the emerging interest in this crop as a source of food ingredients. In this work lupin soluble polysaccharide (LuPS) was obtained with a recovery as high as 46 % by extraction at pH 8, 120 °C, for 90 min. This fraction, named LuPS-8, was composed of a mostly linear pectic polysaccharide with a weight average molecular mass of 6608 kg/mol, and containing 71.0 % galactose, with minor amounts of arabinose (16.0 %), glucuronic acid 4.6 %, and galacturonic acid 4.1 %. When added to an acid milk dispersion, LuPS-8 improved its dispersibility, providing storage stability against sedimentation over a wider pH range than a HM-pectin reference, between 3.6-4.4. This research demonstrated the potential for upcycling of a side stream of lupin protein production, by the creation of value-added novel functional polysaccharide., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Prof. Nakamura has a joint appointment with Fuji Oil Holdings, Japan., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The demand for plant-based protein sources in the food industry has significantly increased in recent years, leading to the introduction of legume-based products as meat substitutes. However, concerns regarding food quality have emerged, particularly related to the presence of mycotoxins. This study addresses the need for the sensitive detection of phomopsins (PHOs), a class of peptide-based toxins. A selective extraction method using molecularly imprinted polymer (MIP) coupled with ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) was focused on the most toxic Phomopsin A (PHO-A). A rapid ultrasonochemical synthesis of MIP (5 min) was proposed and its performance was optimized in response to various factors, including the choice of dummy template and the selection of the monomer. The methacrylic acid-vinyl pyridine (MAA-VP) MIP exhibited high selectivity and affinity for PHO-A. The method was tested in lupin samples and the validation, according to SANTE/11312/2021 international guidelines, gave excellent recovery (80-90 %), low matrix effects, and high accuracy and precision. Real samples analysis confirmed the presence of PHO-A in artificially fungal inoculated lupins, with levels ranging from 0.377 to 0.576 mg kg -1 . In order to identify further PHOs, a semi-untargeted approach using multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) was developed. PHO-B, PHO-D, PHO-E and PHO-P, rarely previously reported in lupin matrix, were tentatively identified. This study accounts for the effectiveness of MIP-based extraction coupled with UHPLC-triple quadrupole with linear ionic trap-MS/MS (UHPLC-QqQ-LIT-MS/MS) for quantification of PHO-A and putative detection of other PHOs, offering a promising method for investigating this class of toxins in food., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Surface tension (γ eq ) of the seed extracts of four lupine cultivars showed values in the range 44.9-46.4 mN/m. The surface compression elasticity (E') of the adsorbed layers and foaming capacity (FC) also showed similar values (E' ∼ 30 mN/m, FC ∼ 100%). The effect of defatting prior to extraction at pH 8.5 depends on the solvent employed - hexane and dichloromethane improved the subsequent protein extraction yield, while ethanol reduced it. The effect of defatting on surface tension could be positive (for hexane and ethanol) or negative (for dichloromethane). Generally, defatting improved the surface compression rheological and foaming parameters. On the other hand, fractionation of the extracts obtained at pH 8.5 from hexane-defatted seeds did not improve significantly the surface activity parameters. Some improvement with respect to the unfractionated extracts was observed only for the extracts of undefatted seeds. γ eq , E', E" and FC isotherms confirm the surfactant-like behavior of the lupine seed extracts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lupines (Lupinus spp.) are a common plant species on western U.S. rangelands with several lupine species containing alkaloids that can be toxic and/or teratogenic to livestock. In North America, more than 150 lupine species are recognized with some ranches or grazing allotments containing multiple species. One or more of these lupine species may contain alkaloids that are teratogenic to cattle. Previous work has shown that lupine alkaloids can be detected in earwax of cattle grazing lupine infested rangelands. Our hypothesis is that earwax can be used to determine if cattle have been exposed to teratogenic alkaloids from multiple lupine species. Two lupine species, L. sericeus and L. polyphyllus, were present on a rangeland in east-central Idaho. The teratogen, anagyrine, was detected in L. sericeus and the teratogen, ammodendrine, was detected in L. polyphyllus plants collected on this rangeland. In this study, earwax was collected from 69 pregnant cows that had previously grazed a rangeland containing two different lupine species containing alkaloids that cause crooked calf syndrome (CCS). Anagyrine was detected in the earwax of all 69 cows sampled. Ammodendrine, was detected in the earwax of 28 of the 69 cows sampled. Earwax is a good non-invasive sample to aid in the diagnosis of cattle that have consumed lupine and does appear, in this case, to be a good diagnostic tool to differentiate between more than one lupine species that may be the cause of CCS. Concentrations of anagyrine or ammodendrine did not correlate with the incidence of CCS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

The effect of the diet modification (soybean and lupine addition) on the content of protein and amino acids (AA) in eggs was studied. Both the sampling day and the diet influenced the total protein content. In albumen, the lowest protein content (10.6%) was noted after administering a diet containing 25% lupine; in the same egg the yolk contained the most proteins (16.7%). In the content of nonessential AA (NAA) in egg yolks, differences were noted only for cysteine, with its the highest content in the yolks of the control group. The stable content of essential yolk amino acids (EAA) was observed only for isoleucine, leucine, tryptophan and phenylalanine. The highest contents of EAA and NAA were recorded in the yolks of the control group (~47 and ~53 g/100 g of protein, respectively) and in the group with 25% additions of lupine (~42 and ~51 g/100 g of protein, respectively). AA with constant content in the tested albumens were methionine, tryptophan and alanine. The highest content of EAA (>~42 g/100 g of protein) and NAA (>~62 g/100 g of protein) were determined in albumen of eggs determined in the group with at least 20% additions of lupine. The highest content of EAA for humans delivered eggs from groups 4-6 (with the addition of soy into the diet ≤5%). The protein sources used in the hen diet significantly influenced the content of protein and individual AA in the produced eggs.

Lupins are used in animal feed because of their excellent nutritional composition. Australian and European Lupinus angustifolius seeds are incorporated in compound feed of calves for veal production in Belgium. To investigate the co-occurrence of quinolizidine alkaloids (QAs) and phomopsin A (PHO A) in lupin seeds and lupin-containing feed, and the potential transfer to animal-derived foods, representative samples were obtained from various actors in the chain. A UHPLC-MS/MS method was validated for the simultaneous quantification of seven QAs and PHO A in relevant matrices. Results indicate highly consistent total QA (TQA) levels in Australian lupins (173 ± 24 mg/kg) ( n  = 25), while European samples showed a high variability (1442 ± 1497 mg/kg) ( n  = 15). PHO A was detected in 7 of 40 samples. Lupin-containing feed had a mean TQA content of 42 ± 28 mg/kg ( n  = 20). An in vivo feeding trial demonstrated the transfer of QAs to muscle and liver of calves that were fed the lupin-containing feed. Highest concentrations were found for lupanine in liver tissue samples (67 ± 46 µg/kg). PHO A concentrations were below the LOD in all feed and tissue samples. These results indicate that animal-derived foods (veal meat/liver) are a potential route for QAs to enter the food chain.

In this work we examined the contents of 14 polyphenols in white lupin ( Lupinus albus L.) samples cultivated in Southern Italy by the optimisation and validation of a LC-HRMS method. The validation of the LC-HRMS method showed linearity results r 2 > 0.989 and recovery values between 71 and 119% for a very wide range of concentrations. Ellagic acid was the most abundant polyphenol, with mean concentrations of 16271.86 ± 19798.53 μg/Kg, followed by apigenin (2749.51 ± 889.95 μg/Kg). A significant variability in ellagic acid contents was found between the areas of cultivation examined ( p  < 0.05). As far as we know, this work provides the first data on the polyphenols contents of white lupins cultivated in Italy. The comparison with other study confirms the role of the cultivation area for the determination of the polyphenol's contents. The study also confirms white lupins as a promising source of antioxidant and anti-inflammatory substances in a balanced diet.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024 The Authors. Ecology published by Wiley Periodicals LLC on behalf of The Ecological Society of America.)

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., (© 2023 Society of Chemical Industry.)

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.

Green synthesis of nanomaterials is advancing due to their ease of synthesis, cheapness, nontoxicity, and renewability. An environmentally friendly biogenic method has been developed for the green synthesis of nickel oxide nanoparticles (NiO NPs) using phytochemical-rich bioextract. They are rich in bioextract phenolics, flavonoids, and berberine. These phytochemicals successfully reduce and stabilize NiNO 3 into NiO NPs. In this study, NiO NPs were synthesized by the green synthesis method from Lupinus Albus. Characterization of NiO NPs was carried out by TEM, XRD, SEM, UV, XRF, BET, and EDX analyses. According to XRD analysis, TEM results also support this, where the NiO NPs particle size diameter is 5 nm. It was determined by the Tauc equation that the band energy gap of NiO NPs is 1.69 eV. It was determined that the BET surface area of NiO NPs was 49.6 m 2 /g. NiO nanoparticles synthesized from Lupinus Albus extract by the green synthesis method were used as catalysts in the photocatalytic reduction of methylene blue with NaBH 4 . In the photocatalytic reduction of methylene blue with NaBH 4 , it was determined that there was no color change in 48 h without a catalyst, and in the presence of NiO nanoparticle catalyst, methylene blue was reduced by 97% in 8 min. The kinetics of the photocatalytic reduction of methylene blue with NaBH 4 is a pseudo-first-order kinetic model and the kinetic rate constant is determined as 0.66 min -1 , indicating that the catalytic effect of NiO NPs is very high at this value. NiO NPs were used five times in the photocatalytic reduction of methylene blue with NaBH 4 and it was determined that the reduction of methylene blue was over 90% in each use.

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 β-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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 β-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.

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) β-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 β-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.

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., (© 2022. The Author(s).)

Covering: up to 2022Quinolizidine alkaloids (QAs) are a class of alkaloids that accumulate in a variety of leguminous plants and have applications in the agricultural, pharmaceutical and chemical industries. QAs are notoriously present in cultivated lupins ( Lupinus spp.) where they complicate the use of the valuable, high-protein beans due to their toxic properties and bitter taste. Compared to many other alkaloid classes, the biosynthesis of QAs is poorly understood, with only the two first pathway enzymes having been discovered so far. In this article, we review the different biosynthetic hypotheses that have been put forth in the literature (1988-2009) and highlight one particular hypothesis (1988) that agrees with the often ignored precursor feeding studies (1964-1994). Our focus is on the biosynthesis of the simple tetracyclic QA (-)-sparteine, from which many of the QAs found in lupins derive. We examine every pathway step on the way to (-)-sparteine and discuss plausible mechanisms, altogether proposing the involvement of 6-9 enzymes. Together with the new resources for gene discovery developed for lupins in the past few years, this review will contribute to the full elucidation of the QA pathway, including the identification and characterization of the missing pathway enzymes.

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.

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 O 2 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.

Background: Basic 7S globulins, a group of proteins commonly found in legumes, undergo the intriguing phenomenon of release from the seeds into hot water. γ-Conglutin is a representative of this group of proteins found in lupin seeds. The physiological significance and the molecular mechanism of the selective release of γ-conglutin from lupin seeds remain unknown. Therefore, the presented study aimed to determine changes in the functionality of this protein in response to the high temperature occurring during lupin seed incubation., Results: It was confirmed that the main protein fraction released from the seeds during high-temperature incubation was γ-conglutin. The incubation condition favours the occurrence of this protein in a monomeric form, and the temperature used corresponds to its midpoint unfolding temperature. Subsequent analysis carried out on the γ-conglutin monomer revealed changes in its functionality after heat shock. The thermally treated protein shows a considerable increase in its interaction strength with flavonoids. Moreover, the inhibitory activity against glycoside hydrolases was enhanced when γ-conglutin monomer was exposed to specific temperatures., Conclusion: The results of the present study provide a potential explanation of the physiological relevance of γ-conglutin and shed new light on a possible mechanism of its activation upon specific heat treatment. This knowledge will help characterise homologous proteins, which are commonly found in other legumes and undergo a similar heat-induced secretion phenomenon. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

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.

Background: This study examined the functional and antioxidant properties of Maillard reaction (MR) products of lupin protein isolate (LPI), fermented (FLPI), and germinated (GLPI) with glucose (G), treated with ultrasound (US) at different power levels (20-40-60-80%) for 15 min. The MR was conducted in a water bath for 180 min at 90 °C., Results: The Trolox-equivalent antioxidant capacity (TEAC) values were found to be 46.79%, 56.43%, and 35.56% for the control (C), 58.99%, 80.17%, and 69.73% for conjugates of LPI-G, FLPI-G, and GLPI-G treated at 80% US, respectively. The maximum 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of LPI-G, FLPI-G, and GLPI-G was found to be 39.68%, 59.54%, and 48.41%, respectively after 80% US. The FLPI-G sample showed the highest antioxidant activity compared with the samples treated at the same power level for DPPH and TEAC. The Fe-chelating activity of GLPI-G showed significant differences when compared with FLPI-G. The solubility of LPI-G, FLPI-G, and GLPI-G increased with increasing US power. The highest solubility was 74.29% for 80% US-treated GLPI-G. The emulsifying activity index (EAI) increased at 20% US and decreased with further increase in the US power. The EAI and emulsifying stability index (ESI) were negatively affected by the MR and US processes., Conclusion: The findings of current study proved that conjugation of LPI with G with the MR and with US pretreatment is an effective method for improving the bio- and techno-functional properties of LPI. It is therefore likely that the properties of plant proteins modified by biochemical and physical treatments may widen their applications in the food industry. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Fusarium oxysporum is an aggressive phytopathogen that affects various plant species, resulting in extensive local and global economic losses. Therefore, the search for competent alternatives is a constant pursuit. Quinolizidine alkaloids (QA) are naturally occurring compounds with diverse biological activities. The structural diversity of quinolizidines is mainly contributed by species of the family Fabaceae, particularly the genus Lupinus . This quinolizidine-based chemo diversity can be explored to find antifungals and even mixtures to address concomitant effects on F. oxysporum. Thus, the antifungal activity of quinolizidine-rich extracts (QREs) from the leaves of eight greenhouse-propagated Lupinus species was evaluated to outline promising QA mixtures against F. oxysporum. Thirteen main compounds were identified and quantified using an external standard. Quantitative analysis revealed different contents per quinolizidine depending on the Lupinus plant, ranging from 0.003 to 32.8 mg/g fresh leaves. Bioautography showed that all extracts were active at the maximum concentration (5 µg/µL). They also exhibited >50% mycelium growth inhibition. All QREs were fungistatic except for the fungicidal QRE of L. polyphyllus Lindl. Angustifoline, matrine, 13α-hydroxylupanine, and 17-oxolupanine were ranked to act jointly against the phytopathogen. Our findings constitute reference information to better understand the antifungal activity of naturally afforded QA mixtures from these globally important plants.

This study focused on the extraction, purification, and physicochemical characterization of γ-conglutin, a protein present in lupin seeds with properties of reducing blood glucose levels. Total protein was extracted with an alkaline-saline solvent, followed by isoelectric precipitation. Chromatographic purification of the precipitated fraction was performed using a cation exchange supermacroporous cryogel column. Electrophoresis of the eluted fraction from chromatography presented a single band of ∼48 kDa under non-reducing conditions (two bands of ∼30 and ∼17 kDa, under reducing conditions) confirming the success of the purification protocol. Liquid chromatography-tandem mass spectrometry analysis confirmed the identity of the protein as γ-conglutin. The purified γ-conglutin had an isoelectric point of 7.51, β-sheets prevailing as a secondary structure, and denaturation temperature close to 68°C. The outcome of this work showed that γ-conglutin was obtained with a high degree of purity. The proposed purification protocol is simple and can be easily scaled up., (© 2021 Wiley-VCH GmbH.)

Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg·kg -1 . After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from δ-conglutin and, to a lower extent, small fragments of β-conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 µg·mL -1 without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system.

Context: Gamma conglutin (Cγ) from lupine species represents a potential complementary treatment for type 2 diabetes mellitus (T2DM) because of its hypoglycaemic effect. However, its underlying mechanism of action is not fully known., Objective: To evaluate whether Cγ from Lupinus rotundiflorus M. E. Jones (Fabaceae) modulates c-Jun N-terminal kinase 1 (JNK1) expression and activation in a T2DM rat model., Materials and Methods: Gamma conglutin isolated from L. rotundiflorus seeds was characterized by SDS-PAGE. Fifteen Wistar rats with streptozotocin-induced T2DM (HG) were randomized into three groups ( n  = 5): vehicle administration (HG-Ctrl), oral treatment with Cγ (120 mg/kg/day) (HG-Lr) for one week, and treatment with metformin (300 mg/kg/day) (HG-Met); a healthy group (Ctrl, n  = 5) was included as control. The levels of glucose and biomarkers of renal and hepatic function were measured pre- and post-treatment. Hepatic Jnk1 expression and phosphorylation of JNK1 were evaluated by qRT-PCR and western blot, respectively., Results: Oral treatment with either Cγ or metformin reduced serum glucose level to 86.30 and 74.80 mg/dL, respectively ( p ˂ 0.05), from the basal levels. Jnk1 expression was 0.65- and 0.54-fold lower ( p ˂ 0.05) in the HG-Lr and HG-Met groups, respectively, than in HG-Ctrl. Treatment with Cγ decreased JNK1 phosphorylation. However, Cγ did not change the levels of kidney and liver biomarkers., Discussion and Conclusions: Treatment with Cγ from L. rotundiflorus inhibited Jnk1 expression, in vivo , suggesting JNK1 as a potential therapeutic target in diabetes and revealing one mechanism underlying the hypoglycaemic effect of lupine Cγ. Nevertheless, further studies are required.

Animal digestive systems host microorganism ecosystems, including integrated bacteria, viruses, fungi, and others, that produce a variety of compounds from different substrates with healthy properties. Among these substrates, α-galacto-oligosaccharides (GOS) are considered prebiotics that promote the grow of gut microbiota with a metabolic output of Short Chain Fatty Acids (SCFAs). In this regard, we evaluated Lupinus albus GOS (LA-GOS) as a natural prebiotic using different animal models. Therefore, the aim of this work was to evaluate the effect of LA-GOS on the gut microbiota, SCFA production, and intestinal health in healthy and induced dysbiosis conditions (an ulcerative colitis (UC) model). Twenty C57BL/6 mice were randomly allocated in four groups ( n = 5/group): untreated and treated non-induced animals, and two groups induced with 2% dextran sulfate sodium to UC with and without LA-GOS administration (2.5 g/kg bw). We found that the UC treated group showed a higher goblet cell number, lower disease activity index, and reduced histopathological damage in comparison to the UC untreated group. In addition, the abundance of positive bacteria to butyryl-CoA transferase in gut microbiota was significantly increased by LA-GOS treatment, in healthy conditions. We measured the SCFA production with significant differences in the butyrate concentration between treated and untreated healthy groups. Finally, the pH level in cecum feces was reduced after LA-GOS treatment. Overall, we point out the in vivo health benefits of LA-GOS administration on the preservation of the intestinal ecosystem and the promotion of SCFA production.

Lupin holds an important place among the legumes and the utilization of lupin as a dietary protein source is an excellent environmentally friendly alternative to animal-based products for human nutrition. In the present study, nutritional, thermal, rheological and functional properties of nine Australian lupin cultivars have been assayed in order to find the most valuable one, both nutritiously and industrially. The set comprised six Lupinus angustifolius L. viz., Barlock, Gunyadi, Jenabillup, Jindalee, Jurien, Mandelup and three Lupinus albus L. viz., Luxor, Rosetta, WK388 cultivars. The tests included analysis of color, macronutrient and micronutrient composition, pasting, textural and thermal properties, electrophoretic profile of protein isolates, swelling power, water and oil absorption capacity, emulsifying capacity, emulsion stability, creaming stability, foaming capacity and stability of the cultivars' dehulled seed flours. The results indicated substantial variation in macro and micro-nutritional value as well as satisfactory swelling ability, solubility, surface hydrophobicity, foaming ability, emulsifying capacity and gelation property of lupin flours. Superior nutritional, thermal, rheological and functional potential was demonstrated by the L. albus cultivars compared to the L. angustifolius cultivars with the exception of Mandelup., (© 2021. The Author(s).)

Agri-food industries generate several by-products, including protein-rich materials currently treated as waste. Lupine species could be a sustainable alternative source of protein compared to other crops such as soybean or chickpea. Protein hydrolysates contain bioactive peptides that may act positively in disease prevention or treatment. Inflammatory responses and oxidative stress underlie many chronic pathologies and natural treatment approaches have gained attention as an alternative to synthetic pharmaceuticals. Recent studies have shown that lupin protein hydrolysates (LPHs) could be an important source of biopeptides, especially since they demonstrate anti-inflammatory properties. However, due to their possible degradation by digestive and brush-border enzymes, it is not clear whether these peptides can resist intestinal absorption and reach the bloodstream, where they may exert their biological effects. In this work, the in vitro cellular uptake/transport and the anti-inflammatory and antioxidant properties of LPH were investigated in a co-culture system with intestinal epithelial Caco-2 cells and THP-1-derived macrophages. The results indicate that the LPH crosses the human intestinal Caco-2 monolayer and exerts anti-inflammatory activity in macrophages located in the basement area by decreasing mRNA levels and the production of pro-inflammatory cytokines. A remarkable reduction in nitric oxide and ROS in the cell-based system by peptides from LPH was also demonstrated. Our preliminary results point to underexplored protein hydrolysates from food production industries as a novel, natural source of high-value-added biopeptides.

Lupin γ-conglutin beneficially modulates glycemia, but whether it protects against oxidative and lipotoxic damage remains unknown. Here, we studied the effects of γ-conglutin on cell death provoked by hydrogen peroxide and palmitate in HepG2 hepatocytes and insulin-producing MIN6 cells, and if a modulation of mitochondrial potential and reactive oxygen species (ROS) levels was involved. We also investigated how γ-conglutin influences insulin secretion and electrical activity of β-cells. The increased apoptosis of HepG2 cells exposed to hydrogen peroxide was prevented by γ-conglutin, and the viability and ROS content in γ-conglutin-treated cells was similar to that of non-exposed cells. Additionally, γ-conglutin partially protected MIN6 cells against hydrogen peroxide-induced death. This was associated with a marked reduction in ROS. No significant changes were found in the mitochondrial potential of γ-conglutin-treated cells. Besides, we observed a partial protection against lipotoxicity only in hepatocytes. Unexpectedly, we found a transient inhibition of insulin secretion, plasma membrane hyperpolarization, and higher K ATP channel currents in β-cells treated with γ-conglutin. Our data show that γ-conglutin protects against cell death induced by oxidative stress or lipotoxicity by decreasing ROS and might also indicate that γ-conglutin promotes a β-cell rest, which could be useful for preventing β-cell exhaustion in chronic hyperglycemia., (Copyright © 2021 Elsevier B.V. All rights reserved.)

There is a renewed interest on the reliance of food-based bioactive compounds as sources of nutritive factors and health-beneficial chemical compounds. Among these food components, several proteins from foods have been shown to promote health and wellness as seen in proteins such as α/γ-conglutins from the seeds of Lupinus species (Lupin) , a genus of leguminous plant that are widely used in traditional medicine for treating chronic diseases. Lupin-derived peptides (LDPs) are increasingly being explored and they have been shown to possess multifunctional health improving properties. This paper discusses the intestinal transport, bioavailability and biological activities of LDPs, focusing on molecular mechanisms of action as reported in in vitro, cell culture, animal and human studies. The potentials of several LDPs to demonstrate multitarget mechanism of regulation of glucose and lipid metabolism, chemo- and osteoprotective properties, and antioxidant and anti-inflammatory activities position LDPs as good candidates for nutraceutical development for the prevention and management of medical conditions whose etiology are multifactorial.

Fermented soy sauces are used as food seasonings in Eastern countries and all over the world. Depending on their cultural origins, their production differs in parameters such as wheat addition, temperature, and salt concentration. The fermentation of lupine seeds presents an alternative to the use of soybeans; however, the microbiota and influencing factors are currently unknown. In this study, we analyse the microbiota of lupine Moromi (mash) fermentations for a period of six months and determine the influence of different salt concentrations on the microbiota dynamics and the volatile compound composition. Cultured microorganisms were identified by protein profiling using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene amplicon sequencing provided an overview of the microbiota including non-cultured bacteria. The volatile compounds were determined by gas chromatography-mass spectrometry (GC-MS). At all salt concentrations, we found that Tetragenococcus halophilus (up to 1.4 × 10 9 colony forming units (CFU)/mL on day 21) and Chromohalobacter japonicus (1.9 × 10 9  CFU/mL, day 28) were the dominating bacteria during Moromi fermentation. Debaryomyces hansenii (3.6 × 10 8  CFU/mL, day 42) and Candida guilliermondii (2.2 × 10 8  CFU/mL, day 2) were found to be the most prevalent yeast species. Interestingly, Zygosaccharomyces rouxii and other yeasts described as typical for soy Moromi were not found. With increasing salinity, we found lower diversity in the microbiota, the prevalence-gain of typical species was delayed, and ratios differed depending on their halo- or acid tolerance. GC-MS analysis revealed aroma-active compounds, such as pyrazines, acids, and some furanones, which were mostly different from the aroma compounds found in soy sauce. The absence of wheat may have caused a change in yeast microbiota, and the use of lupine seeds may have led to the differing aromatic composition. Salt reduction resulted in a more complex microbiome, higher cell counts, and did not show any spoiling organisms. With these findings, we show that seasoning sauce that uses lupine seeds as the sole substrate is a suitable gluten-free, soy-free and salt reduced alternative to common soy sauces with a unique flavour., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Scope: We have previously demonstrated the anti-inflammatory and antioxidant properties of in vitro administered Lupinus angustifolius protein hydrolysates (LPHs) on human peripheral blood mononuclear cells (PBMCs). This study aims to evaluate the safety and efficacy of a beverage containing LPHs (LPHb) on the immune, oxidative and metabolic status of healthy subjects., Methods and Results: In this open-label intervention, 33 participants daily ingest a LPHb containing 1 g LPHs for 28 days. Biochemical parameters are assayed in fasting peripheral blood and urine samples before, during (14 days) and after LPHb ingestion. Participants' health status and the immune and antioxidant responses of PBMCs are also evaluated throughout the trial. The LPHb ingestion is safe and effective in both increasing the anti-/pro-inflammatory response of PBMCs and improving the cellular anti-oxidant capacity. LPHb also reduces the low-density lipoprotein-cholesterol (LDL-C)/high-density lipoprotein-cholesterol (HDL-C) atherogenic index. LPHb effect is particularly beneficial on decreasing not only the LDL-C/HDL-C index but also serum total cholesterol levels in the male cohort that shows the highest baseline levels of well-known cardiovascular risk factors., Conclusion: This is the first study to show the pleiotropic actions of a lupine bioactive peptides-based functional food on key steps of atherosclerosis including inflammation, oxidative stress, and cholesterol metabolism., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)