One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.     

Nitrogen fractions and mineral content in different lupin species (Lupinus albus, Lupinus angustifolius, and Lupinus luteus). Changes induced by the alpha-galactoside extraction process

The protein and mineral composition of different varieties of three different lupin species (Lupinus albus, Lupinus angustifolius, and Lupinus luteus) and the effect of alpha-galactoside removal by means of a hydroalcoholic extraction process on such composition were studied in relationship to nutrient distribution among the different anatomical parts of the seed (embryo, cotyledon, and seed coat). The extent of processing-derived protein insolubilization was assessed by both chemical and electrophoretic techniques and related to the amount of nitrogen soluble in H(2)O, NaCl, ethanol, NaOH, and sodium dodecyl sulfate/beta-mercaptoethanol (SDS/BME). The alpha-galactoside extraction process caused a significant increase in the amount of total and insoluble nitrogen and decreased the amount of soluble protein nitrogen, without affecting the content of soluble nonprotein nitrogen. alpha-Galactoside extraction was not effective at decreasing the levels of Mn present in lupins, and processing caused an increase in the content of this mineral in all of the species studied with the exception of L. albus var. multolupa. In general, the effect of processing on mineral content varied with the different lupin species, and mineral losses were lower in L. luteus.     

Influence of lupin (Lupinus luteus L. cv. 4492 and Lupinus angustifolius L. var. zapaton) and fenugreek (Trigonella foenum-graecum L.) germination on microbial population and biogenic amines

Microbial population and bioactive amine profile and levels of two lupin species (Lupinus luteus L. cv. 4492 and Lupinus angustifolius L. var. zapaton) and fenugreek (Trigonella foenum-graecum L.) seeds as affected by germination were investigated. Microbial population increased considerably mainly in the first stage of germination (2 days), then small changes in bacterial numbers were observed up to 5 days to levels between 7.8 and 8.9 log colony-forming units/g. Microorganisms belonging to the Enterobacteriaceae family were dominant for the legumes tested. Ungerminated legume seeds contained putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. Bioactive amine levels found in fenugreek seeds were between 3- and 4-fold higher than those found in lupin seeds. The highest total amine levels were found in fenugreek seeds [162 mg/kg of dry weight (dw)], followed by L. angustifolius var. zapaton seeds (84 mg/kg of dw) and, finally, L. luteus cv. 4492 (46 mg/kg of dw) seeds. The concentration of individual amines showed a gradual rising trend during the germination period in all tested sprouts, reaching levels >3 times higher than those found in ungerminated seeds. After 5 days of germination, the fenugreek sprouts contained the highest amount of total bioactive amines. Tyramine was the predominant amine in both lupin varieties, whereas cadaverine was the main bioactive amine detected in fenugreek. The results of this work thus indicated that microbial population and biogenic amine levels in the studied lupin and fenugreek sprouts are not a risk for healthy consumers or for individuals with restricted activity of detoxification enzymes.     

Identification and quantification of epsilon-(gamma-glutamyl)lysine in digests of enzymatically cross-linked leguminous proteins by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS)

A rapid and convenient method for the precise quantification of epsilon-(gamma-glutamyl)lysine isopeptide in lyophilized proteolytic digests of cross-linked plant protein samples was developed. The isopeptide was baseline-separated from three other isomers containing lysyl and glutamyl residues by reverse-phase high-performance liquid chromatography after exhaustive proteolytic digestion of the samples cross-linked by a microbial transglutaminase (MTG). Highly selective detection was performed by electrospray mass spectrometry in MS/MS mode. Demonstrating the applicability of the suggested analytical procedure, enzymatic cross-linking of protein isolates from soy [Glycine max (L.) Merr.], pea [Pisum sativum L.], and the sweet lupin species Lupinus albus L. and Lupinus angustifolius L. was investigated after incubation with 0.01 g of MTG/100 g of protein for 0-240 min at 40 degrees C. The liquid chromatography-mass spectrometry (LC-MS) method was successfully applied to monitor the kinetics of epsilon-(gamma-glutamyl)lysine isopeptide formation. Since the calculated initial levels of epsilon-(gamma-glutamyl)lysine in the genuine leguminous protein isolates were between 40 and 77 micromol/100 g, an isopeptide detection limit of 0.5 microg/mL, corresponding to approximately 50 micromol/100 g of protein, was shown to suffice for quantifying the cross-linking rate enzymatically induced by MTG. Concentrations of epsilon-(gamma-glutamyl)lysine in the texturized proteins ranged from 100 to 500 micromol/100 g of protein.     

Comparative phosphorus requirement of four lupin species

The yield response of yellow lupin (Lupinus luteus), sand plain lupin (L. cosentinii), narrow‐leafed lupin (L. angustifolius), and white lupin (L. albus) to applications of phosphorus (P), as single superphosphate (0 to 80 kg P ha^‐1), was measured in the year of application in a field experiment on a sandy soil. Comparative P requirement was determined from yields when no P was applied, the amount of P required to produce the same percentage of the maximum (relative) yield, such as 90% of the maximum yield, and the P content measured in dried tops. For all these criteria, P requirement generally increased in the order L. cosentinii < L. albus < L. angustifolius < L. luteus. Proteoid roots, found only on L. cosentinii and L. albus plants, were probably responsible for these species using indigenous soil P and applied fertilizer P more effectively than L. luteus and L. angustifolius.     

Extraction, isolation, and characterization of globulin proteins from Lupinus albus

Lupin has recently been added to the list of allergens requiring mandatory advisory labeling on foodstuffs sold in the European Union, and since December 2008, all products containing even trace amounts of lupin must be labeled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and "legumin-like") and β-conglutin (7S and "vicilin-like") and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. We report on a methodology to facilitate the extraction of each of these proteins using centrifugation and isolation by anion-exchange chromatography followed by size-exclusion chromatography. The isolated subunits were characterized using reducing and non-reducing polyacrylamide gel electrophoresis, western blotting, and peptide mass fingerprinting, all of which revealed that the individual protein subunits are highly pure and can be used as immunogens for the production of antibodies specific for each of the conglutin fractions, as well as standards, and the extraction protocol can be used for the selective extraction of each of the subunits from foodstuffs, thus facilitating a highly accurate determination of the lupin concentration. Furthermore, the subunits can be used to elucidate information regarding the toxicity of each of the subunits, by looking at their interaction with the IgE antibodies found in the serum of individuals allergic to lupin, providing critical information for the definition of the requirements of analytical assays for the detection of lupin in foodstuffs.     

Biological activity of alpha-galactoside preparations from Lupinus angustifolius L. and Pisum sativum L. seeds.

Biological activity tests were performed on alpha-galactoside preparations obtained from Lupinus angustifolius L. cv. Mirela (alkaloid-rich) and Pisum sativum L. cv. Opal seeds. The studies included the following tests: acute toxicity, cytotoxic test, delayed type hypersensitivity (DTH), plaque-forming cell number (IgM-PFC), and influence on the growth of bifidobacteria and coliform presence in rat colon. Results of these studies showed that alpha-galactosides from lupin and pea seeds were essentially nontoxic. Their acute toxicity (LD(50)) in mice was >4000 mg kg(-1) of body weight. alpha-galactoside preparations were not cytotoxic for mouse thymocytes in vitro. The in vitro test shows that oligosaccharides from lupin and pea are utilized by selected beneficial colon bacterium strains. The in vivo experiment demonstrated that alpha-galactosides from legume significantly influenced the growth of bifidobacteria in rats colon. Simultaneously, the decrease of the coliform presence was observed. The chemical composition of the tested preparations had no significant effect on their biological activity.     

Metal ions restore the proteolytic resistance of denatured conglutin gamma, a lupin seed glycoprotein, by promoting its refolding

The susceptibility to trypsin of conglutin gamma, a lupin seed glycoprotein affected by this enzyme only when in a non-native conformation, was used to study the effect of Zn(2+) and other metal ions on the structural dynamics of the protein. When acid-treated trypsin-susceptible conglutin gamma was incubated at neutral pH in the presence of Zn(2+), it became resistant to tryptic attack, contrary to the protein treated in the absence of Zn(2+). The time course of this refolding event has been quantitatively evaluated by SDS-PAGE. Amino acid sequencing of the major polypeptide fragments, produced by trypsin before completion of the refolding process, indicated that only a few cleavable bonds were accessible to the enzyme. This suggested that the presence of metal ions affected the pathway of degradation of the protein, by inducing its folding. Among the other metal ions tested, Ni(2+) also promoted the adoption of a trypsin-resistant conformation of conglutin gamma, whereas Mn(2+) and Ca(2+) had only much lower effects. The relevance of these findings for a deeper understanding of the in vivo degradation of plant food proteins and how it is affected by metal ions are discussed.     

Quinolizidine alkaloids in seeds of lupin genotypes of different origins

The intake of lupin-based foods could imply the exposure of consumers to quinolizidine alkaloids. The objectives of this study were to assess the genetic variation among and within 11 geographic regions of Lupinus albus ecotypes, verify the quinolizidine alkaloids amount of alkaloid-poor L. albus and Lupinus angustifolius varieties, and assess the effect of two climatically contrasting Italian environments on the alkaloid content. The quantitation was performed by GC-MS, and in all samples lupanine was the most abundant quinolizidine alkaloid, followed by albine and 13alpha-hydroxylupanine for L. albus and by 13alpha-hydroxylupanine and angustifoline for L. angustifolius. Some regions tended to have a high (Azores) or low (Egypt, Near East, Maghreb) total alkaloids content, but the variation among ecotypes within regions was larger than that among regions following the estimation of variance components. Alkaloid-poor varieties tended to have higher total alkaloid contents when grown in the subcontinental climate site, exceeding in some cases the limit of 0.200 mg/g.     

COMPARING FERTILIZER PHOSPHORUS REQUIREMENTS OF CANOLA,LUPIN, AND WHEAT

Increases in yield due to applications of phosphorus (P) (0, 5, 10, 15, 20, and 40 kg P/ha) applied as single (ordinary) superphosphate were measured for canola (Brassica napus), lupin (Lupinus angustifolius) and wheat (Triticum aestivum) in a field experiment on a deep sandy soil near Esperance, south-western Australia (WA). There are no data comparing the P requirements of these species grown at the same time, which was done by determining the amount of P required to produce 90% of the maximum yield for each species. The amount of P required was about 50% less for canola than wheat and about 10% more for lupin than wheat (60% more than canola). For each amount of P applied, the concentration of P in shoots and grain was greater for canola, followed by lupin and then wheat, suggesting that canola and lupin roots were better at accessing soil P than wheat. The critical concentration of P (diagnostic) required for 90% maximum yield of dried shoots measured in September was about 2.3 g/kg P for wheat, 2.8 g/kg P for lupin, and 3 g/kg P for canola. Similar critical values were obtained when P concentration in the shoots was related to grain yield (prognostic).     

Nitrogen uptake in organically managed spring sown lupins and residual effects on leaching and yield of a following winter cereal

Three unfertilized spring sown species of lupins (Lupinus angustifolius) and peas (Pisum sativa) were compared in terms of N fixation and subsequent leaching under a following winter barley crop in an organically managed rotation. Fallow plots were included to assess the potential weed burden and the ability of the sown crops to compete with weeds when no herbicides were applied. Although peas out-yielded lupins (5.4 t compared with 3.5 t grain respectively), the yellow lupin (Wodjil) fixed more N than peas (180 compared with 120 kg N/ha) and all three lupins had higher protein contents (>30%) than peas (22%). Winter leaching amounted to >50 kg nitrate-N/ha from winter barley, regardless of whether it followed treatments which were previously fallow or cropped with legumes. There were no significant differences in leaching between the lupin species. Leachate in the first 350 mm of drainage under winter barley, following the different legume species, exceeded the European Union limit for nitrate in drinking water in all treatments. Yields of winter barley, grown without fertilizers or herbicides following legumes, were not significantly different (mean 4 t/ha), but there were higher levels of P and K in the grain compared with the amounts made available from the previously fallow soil. At this site in SW England, the crops grew well and our results suggest that lupins could provide a useful break crop in an organic arable cropping rotation and an alternative source of home-grown, high protein feed.     

Thermal stabilities of lupin seed conglutin gamma protomers and tetramers

Various experimental approaches have been used in this work to assess the thermal stabilities of lupin seed conglutin gamma at two pH values, 4.5 and 7.5, at which the protein exists as a protomer and a tetramer, respectively. The patterns of thermal unfolding at the two pH values differed significantly; the tetramer aggregated and became insoluble, whereas the protomer was still soluble after thermal treatment. Also, the midpoint transition temperatures were dramatically different, being 60.3 and 75.1 degrees C for the protomer and tetramer, respectively. The behavior of conglutin gamma at neutral pH was also affected by disulfide formation/interchange, in that some unfolded protein molecules became covalently stabilized. More detailed analyses by differential scanning calorimetry and indirect fluorescence measurements, using 8-anilino-1-naphthalenesulfonic acid as a probe, confirmed the remarkable differences observed in the thermal stabilities of the two protein forms and allowed models for their unfolding patterns to be drawn.     

Are mineral nutrients a critical factor for lime intolerance of lupins?

Poor growth of lupins on calcareous soils may be attributed to a number of soil physical and chemical factors. Nutrient imbalances, such as deficiency of phosphorus (P) and micronutrients or calcium (Ca) excess have been reported to be responsible for the calcifuge behavior of the plants. In the present study we investigated the importance of nutrient imbalances for the growth reduction of lupins on a lime‐containing soil. Three lupin species (Lupinus luteus, Lupinus angustifolius, and Lupinus albus) were compared with lime‐tolerant Pisum sativum. Plants were cultivated in a sandy soil containing 0.2% or 10% magnesium (Mg) limestone and were fertilized with a complete nutrient solution except for iron (Fe). In each lime treatment, three of six pots per species were supplied with iron as FeEDDHA. Strong liming greatly decreased shoot growth, rate of leaf appearance, and shoot dry matter accumulation in all Lupinus species, but only marginally in P. sativum. All Lupinus species displayed chlorosis on the strongly limed soil, whereas on the slightly limed soil, only L. luteus did so. Shoot concentrations of P, potassium (K), Ca, Mg, manganese (Mn), zinc (Zn), and copper (Cu) were generally in the adequate range. Decreased shoot growth was not associated with increased Ca concentrations. FeEDDHA fertilization alleviated chlorosis in most cases, but was not able to restore shoot growth. Therefore it is concluded, that, at least in the juvenile stage, nutrient imbalances do not play a major role in growth limitation of lupins on calcareous soils.     

Lupin Flours as Additives: Dough Mixing,Breadmaking, Emulsifying,and Foaming

The nutritional quality of various food products could be improved by supplementation with grain legumes to increase protein content and to improve the balance of essential amino acids. The lupin grain is a good candidate for this role, given its yield potential in a range of climatic environments and soil types. To establish the practicality of extending the use of lupins as food additives, the functional properties of various species and cultivars of lupin were studied for their effect as additives to baked products and their ability to provide foaming and emulsifying properties. Of the two lupin species that are commonly cultivated commercially, Lupinus albus showed the greater potential as a bread additive; loaf height and structure were maintained when lupin flour was substituted for wheat flour at levels up to 5%. This level of substitution offered the advantage of reducing mixing time. The detrimental effects at higher substitution levels appeared to be associated with the nonprotein components of the lupin flour. L. albus showed better functionality than L. angustifolius in emulsifying attributes, although L. angustifolius showed greater potential as a foaming agent. Defatting the lupin flour may be necessary to show these properties to best advantage. Certain cultivars (within each species) showed preferable performance, indicating the potential for plant breeding to provide germplasm better suited to uses as food additives.     

Potential of landrace germplasm for genetic enhancement of white lupin in Egypt

Landraces of white lupin (Lupinus albus L.) collected in Egypt were evaluated along with locally developed cultivars and selected foreign germplasm for yield and major morphological characteristics in five different locations. These locations represent different soil types and climatic conditions in Egypt. The results showed an outstanding performance of the local cultivar checks across traditional locations for lupin cultivation, which supports the utilization of local germplasm for further improvement of locally adapted lupins in Egypt. The results further suggest that local landrace germplasm may be an important source of alleles for shortening the vegetative period, reducing plant height and stem length, as well as for improving some yield components as number of pods and seeds per plant. The genotype-by-location interaction was significant for all characteristics. Mass selection in the Egyptian germplasm collection of white lupin has the potential for enhancing yield, especially in germplasm adapted to newly reclaimed desert locations.     

Lupinus anatolicus — a new lupin species of the old world

Summary During a collecting mission in South-West Turkey some lupin plants differing from Lupinus pilosus Murr., L. micranthus Guss. and L. angustifolius L., wild-growing in this region were found. These plants markedly distinguished from a dwarf habit of L. micranthus and exuberant L. pilosus. As found later, these plants with regard to many traits differed still more from the remaining lupin species of the Old World. The collected seeds of these plants were multiplied at the Plant Breeding Station in Wiatrowo. They were characterized by a smooth seed coat and according to classification of Gladstones could be referred to a group of European lupin crops containing the primitive species L. micranthus.New accession was compared to L. micranthus and L. pilosus considering 20 morphological, some physiological features and chemotaxonomic analyses. New accession was also artificially crossed to L. micranthus and L. pilosus but hybrid seeds were not obtained. The most pronounced morphological differences were in the height and exuberance of plants, the size of inflorescences and flowers, the size and coloration of strongly pubescent, ripen pods but first of all, the size and shape of seeds and seed coat surface.Differences in the protein and fat content in seeds as well as those in quantitative and qualitative composition of alkaloids also appear to be significant.Marked differences occurred also in the electrophoretic phenotype of isozymes. They consisted not only in differences of electrophoretic mobility of bands, but also in different number of bands.The obtained results enabled us to give the population of these plants a separate species name — Lupinus anatolicus.     

Growth,P uptake in grain legumes and changes in rhizosphere soil P pools

In soils with low P availability, several legumes have been shown to mobilise less labile P pools and a greater capacity to take up P than cereals. But there is little information about the size of various soil P pools in the rhizosphere of legumes in soil fertilised with P although P fertiliser is often added to legumes to improve N₂ fixation. The aim of this study was to compare the growth, P uptake and the changes in rhizosphere soil P pools in five grain legumes in a soil with added P. Nodulated chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), white lupin (Lupinus albus L.), yellow lupin (Lupinus luteus L.) and narrow-leafed lupin (Lupinus angustifolius L.) were grown in a loamy sand soil low in available P to which 80 mg P kg⁻¹ was added and harvested at flowering and maturity. At maturity, growth and P uptake decreased in the following order: faba bean > chickpea > narrow-leafed lupin > yellow lupin > white lupin. Compared to the unplanted soil, the depletion of labile P pools (resin P and NaHCO₃-P inorganic) was greatest in the rhizosphere of faba bean (54% and 39%). Of the less labile P pools, NaOH-P inorganic was depleted in the rhizosphere of faba bean while NaOH-P organic and residual P were most strongly depleted in the rhizosphere of white lupin. The results suggest that even in the presence of labile P, less labile P pools may be depleted in the rhizosphere of some legumes.     

Root Morphology,Proton Release,and Carboxylate Exudation in Lupin in Response to Phosphorus Deficiency

Narrow-leafed lupin (Lupinus angustifolius L.) is widely planted in infertile acidic soils where phosphorus (P) deficiency is one of the major limiting factors for plant growth. A hydroponic experiment was conducted to examine the morphological and physiological responses of roots of narrow-leafed lupin in response to altered P supply at 0, 1, 10, 25 or 75 μ M P as monopotassium phosphate (KH₂PO₄). Low P (P₀ and P₁) significantly decreased the plant biomass, but the supply of 10 μ M P was sufficient to produce similar plant biomass as the maximal P supply (P₇₅), indicating an efficient P acquisition by narrow-leafed lupin. Phosphorus deficiency did not enhance rates of carboxylate exudation and proton release by plant roots, indicating that carboxylate exudation and proton release are not the mechanisms for efficient P acquisition. In contrast, low P supply evidently modified the root morphology by increasing the primary root elongation, and developing a large number of cluster-like first-order lateral roots with dense root hairs, thus allowing efficient P acquisition by narrow-leafed lupin under low P supply.     

Phosphatversorgung von Sommerweizen (Triticum aestivum L.) in Mischkultur mit Weißer Lupine (Lupinus albus L.)

Phosphorus nutrition of spring wheat (Triticum aestivum L.) in mixed culture with white lupin (Lupinus albus L.). Spring wheat (Triticum aestivum L. ?Schirokko”?) and white lupin (Lupinus albus L.) were grown in mixed culture in Mitscherlich pots with 20 kg of soil in a green house. The soil used was a B_t of a Parabraunerde-Pseudogley from loess low in available P and limed from pH 4.6 to pH 6.5. Phosphorus was added as phosphate rock. In half of the pots cylinders of stainless steel screen prevented intertwining of the roots of the plant species. Independent of P addition, white lupin had higher dry matter production and P uptake than wheat, even although wheat had thinner roots and higher root densities than lupin, factors which favour the utilization of soil and fertilizer P. The higher P efficiency of white lupin was due to higher P uptake rates per unit root length mainly through mobilization of P especially in the rhizosphere of the proteoid roots. When the roots of the two species were allowed to intertwine, shoot dry matter production of wheat was nearly double because of improved tillering. Higher P concentrations and a more than 2-fold higher P uptake indicated that the increase in dry matter production of wheat was due to improved P nutrition. Nitrogen concentrations, however, remained unaffected at sufficient levels. An increased P uptake rate per unit root length was responsible for the better utilization of P by wheat, rather than the increase in total root length, due to the extended root volume. White lupin was able to mobilize P in the rhizosphere in excess of its own requirements. Thus mobilized P may be available to less P-efficient plants grown in mixed culture.