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.     

Proteomic analysis of lupin seed proteins to identify conglutin Beta as an allergen, Lup an 1

Lupin products may be valuable as human foods because of their high protein content and potential anticholesterolemic properties. However, a small percentage of the population is allergic to lupin. In this study, we use in vitro IgE binding and mass spectrometry to identify conglutin beta, a major storage protein, as an allergen in seeds of Lupinus angustifolius and Lupinus albus. Purification of conglutin beta from L. angustifolius flour confirmed that serum IgE binds to this protein. Where IgE in sera recognized lupin proteins on Western blots, it recognized conglutin beta, suggesting this protein is a major allergen for lupin. The L. angustifolius conglutin beta allergen has been designated Lup an 1 by the International Union of Immunological Societies (IUIS) allergen nomenclature subcommittee.     

Bacterial removal of quinolizidine alkaloids and other carbon sources from a Lupinus albus aqueous extract

Two Gram-negative bacterial strains capable of using lupanine, the predominant quinolizidine alkaloid in Lupinus albus, as a sole carbon source were isolated from soil in which L. albus and L. luteus had been grown [Santana, F. M. et al. J. Ind. Microbiol. 1996, 17, 110-115]. In the present study, we present results suggesting that these isolates are of potential interest for removing lupanine and other quinolizidine alkaloids (QA) from the effluent resulting from the wet processing of Lupinus seeds, at temperatures within the range 20-34 degrees C. Growth in L. albus aqueous extract was diauxic, with a first period of rapid growth leading to the simultaneous consumption of a significant part of the initial concentration of QA (3 g L(-1), being 2 g L(-1) lupanine) and amino acids (1.5 g L(-1)). This period was followed by a second period of slower growth corresponding to the subsequent partial utilization (25%) of the carbohydrates (initial concentration of 20 g L(-1)) together with further removal of QA and amino acids. Despite the differences detected in the susceptibility of the two strains to lupanine toxicity, in particular at supraoptimal temperatures, and in the efficiency of lupanine catabolism, their performance on L. albus extract did not vary significantly.     

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.     

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.     

Compositional variations for alpha-galactosides in different species of leguminosae, brassicaceae, and barley: a chemotaxonomic study based on chemometrics and high-performance capillary electrophoresis

The contents of raffinose family oligosaccharides (RFO) and sucrose in Brassica, Lupinus, Pisum, and Hordeum species were investigated by chemometric principal component analysis (PCA). Hordeum samples contained sucrose and raffinose, and Brassica samples all contained sucrose, raffinose, and stachyose. In addition to these, the Pisum samples contained verbascose and the Lupinus samples also contained ajugose. High stachyose and low ajugose contents were found in Lupinus albus in contrast to Lupinus angustifolius, having low stachyose and high ajugose contents. Lupinus luteus had average stachyose and ajugose contents, whereas large amounts of verbascose were accumulated in these seeds. Lupinus mutabilis had high stachyose and low ajugose contents, similar to the composition in L. albus but showing higher raffinose content. The Brassica samples also showed compositional RFO variations within the species, and subgroup formations were discovered within the investigated Brassica napus varieties. PCA results indicated compositional variations between the investigated genera and within the various species of value as chemotaxonomic defined parameters and as tools in evaluations of authenticity/falsifications when RFO-containing plants are used as, for example, feed and food additives.     

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.     

The acquisition of cadmium by Lupinus albus L., Lupinus angustifolius L., and Lolium multiflorum Lam.

Das Cadmiumaneignungsvermögen von Lupinus albus L., Lupinus angustifolius L. und Lolium multiflorum Lam. Mehrere Pflanzenarten mobilisieren Bodenphosphate (P) und Kationen wie Fe und Al durch die Exsudation organischer Anionen und Protonen. Deshalb untersuchten wir das Cd‐Aneignungsver‐mögen von P‐, Fe‐, Al‐mobilisierenden Arten (Lupinus albus L., Lupinus angustifolius L.) im Vergleich zu einer nicht mobili‐sierenden Pflanzenart (Lolium multiflorum Lam.). Die Pflanzen wuchsen in zwei stark unterschiedlichen Böden (saurer Humuspodsol, karbonathaltiger Lössunterboden). Die Cd‐Aufnahme in die Sprosse war bei Weidelgras 5 bis 10 mal höher als bei Blauen bzw. Weißen Lupinen. Dieses Ergebnis hat mehrere Ursachen: 1. Das Wurzellängen/Sprossmasseverhältnis des Weidelgrases ist 2—3 mal größer als das der Lupinenpflanzen. 2. Bei Weidelgras wird ein größerer Teil des aufgenommenen Cd in die Sprosse verlagert. 3. Die Cd‐Aufnahme bei Lupinen ist im sauren Boden (Podsol) und bei P‐Mangel auch im Kalkboden niedriger als bei Weidelgras. Während im Podsol die Cd‐Konzentration der Bodenlösung unter Lupine geringer war als in der Kontrolle (Gefäße ohne Pflanzen), war sie im Kalkboden höher. Bei den Lupinen war der Efflux organischer Säureanionen, vor allem Citrat und Malat, um den Faktor 10—100 höher als bei Weidelgras. Diese Exsudation kann zu einer hohen Cd‐Komplexierung, insbesondere durch Citrat, in der Rhizosphärenbodenlösung führen (˜ 85%). Diese Ergebenisse deuten darauf hin, dass das komplexierte Cd von den Wurzeln schlechter aufgenommen wird als das freie Cd.     

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.     

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.     

Novel polyclonal-monoclonal-based ELISA utilized to examine lupine (Lupinus species) content in food products

Sweet lupines are increasingly used in food production. Cause for concern has been expressed due to the increase in reported lupine-induced allergic incidents and the association between lupine and peanut allergies. In the current study, a polyclonal-monoclonal antibody-based sandwich ELISA for the detection of lupine proteins in foods was developed. The assay was sensitive to both native and processed proteins from Lupinus angustifolius and Lupinus albus and had a detection limit of 1 mug/g. Intra- and interassay coefficients of variation were <5 and <17%, respectively. A selection of 112 food samples, both with and without lupine declaration, was evaluated for their content of lupine. The data showed that the majority were in agreement with the respective labeling. However, some inconsistency was seen, typically in bread/rolls and soy flours.     

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.     

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.     

Nutritional potential of raw and free alpha-galactosides lupin (Lupinus albus Var. multolupa) seed flours. Effect of phytase treatment on nitrogen and mineral dialyzability

The effect of the removal of alpha-galactosides from Lupinus albus L. var. multolupa on the chemical composition of the prepared flour and the dialyzability of N, total P, Ca, Mg, Fe, Zn, and Mn was studied. The extraction process caused a significant increase in total and insoluble nitrogen contents and decreased the amount of soluble protein nitrogen. However, neither these changes nor treatment with phytase seemed to considerably affect in vitro protein digestibility. Except for Ca and Cu, total mineral contents were significantly reduced by the extraction process. The process also caused a significant reduction in the dialyzability of all the minerals studied except P. The decrease in mineral dialyzability was partially counteracted by phytase treatment in amounts of 250-500 phytase units/kg of lupin flour. In the case of Fe, mineral dialyzability did not differ significantly between the two lupin flours studied with treatment with 500 phytase units/kg. Zinc dialyzability was the most efficiently improved by phytase treatment (P < 0.0001), followed by P, Fe, and Mn, and finally by Ca and Mg (P < 0.05).     

Nodulation and nitrogen fixation of Lupinus species with Bradyrhizobium (lupin) strains in iron-deficient soil

 The effect of six Bradyrhizobium sp. (lupin) strains (WPBS 3201D, WPBS 3211D, USDA 3040, USDA 3041, USDA 3042 and CB 2272) and Fe supply on nodulation, N₂-fixation and growth of three lupin species (Lupinus termis, L. albus and L. triticale) grown under Fe deficiency in an alkaline soil, were examined in sterilized and non-sterilized pot experiments. When inoculated with USDA 3040, 3041, 3042 and CB2272 without Fe addition, the three lupin species had a very low nodule number and mass, low shoot and root dry matter accumulation and lower N yield. However, inoculation with WPBS 3201D and 3211D without Fe treatments increased all these parameters substantially. The ability of WPBS 3201D and 3211D to form nodules on the three lupin species under conditions of Fe stress could be attributed to their ability to scavenge Fe from Fe-deficient environments through their siderophore production. Addition of Fe to the other four strains significantly increased nodulation and N₂-fixation of the three lupin species, indicating that the poorer nodulation and N₂-fixation of these strains in the absence of Fe, resulted from a low ability to obtain Fe from alkaline soils. Bradyrhizobium strains WPBS 3201D and 3211D were superior to the other four strains in terms of promoting greater nodulation, N₂-fixation, plant growth and N accumulation of L. termis and L. albus. However, the other four strains were more efficient in symbiotic association with L. triticale. The greater variations in nodule efficiencies (specific nitrogenase activity) under different levels of Fe supply could be attributed to the quantities of bacteroid protein and leghaemoglobin in the nodules. The results suggested that Bradyrhizobium (lupin) strains differ greatly in their ability to obtain Fe from alkaline soils, and that the selection of bradyrhizobial strains which are tolerant of Fe deficient soils could complement plant breeding for the selection of legume crops for Fe-deficient soils. Received: 5 January 1998     

Electrophoretic seed globulin patterns in some New World Lupinus species

Comparative SDS-PAGE analysis of seed globulins covered 74 accessions representing 19 New World Lupinus species, 18 North American lupins and one South American species: L. mutabilis. The species investigated showed major, well-defined polypeptide bands in the approximate M _r range of 40–75 kDa. In general, intraspecific variation blurred interspecific differences; L. mutabilis could not be distinguished from the North American lupins. Of the taxa investigated, only L. subcarnosus and L. succulentus displayed some species-specific features. The American species examined showed different seed globulin patterns from those reported earlier for the smooth-seeded and for the rough-seeded Old World lupins.     

Nitrogen turnover of legume seed meals as affected by seed meal texture and quality at different temperatures

The aim was to investigate how legume seed meal texture and corresponding quality affects N turnover at different temperatures. Therefore, the effect of size fractionation ‘fine’ and ‘coarse’ of seed meals of yellow lupin (Lupinus luteus L.), blue lupin (Lupinus angustifolius L.) and faba bean (Vicia faba L.) on net N mineralization and turnover was investigated in an incubation experiment at 5°C, 12°C and 20°C. The differences in N release from the two particle size fractions could not be detected at 12 and 20°C incubation temperature. Moreover, net N mineralization at 5°C was higher during incubation of the coarse particle size fractions than during incubation of the fine fraction. In contrast to the common understanding of temperature dependence of microbial processes, the overall influence of incubation temperature on net N mineralization was less expressed. The formation of microbial biomass was highest at 5°C. The subsequent decrease of soil microbial biomass was only partly reflected by net N mineralization suggesting the formation of microbial residues as a preliminary N sink. The control of the N release from legume seed meals seems to be dominated by the N-immobilizing effects of polyphenols at lower temperatures and of C-rich polymers (hemicelluloses) at higher temperatures.     

Changes of isoflavones during the growth cycle of Lupinus albus

The objective of this study was to monitor the changes in isoflavone content in different plant organs (leaves, stems, roots) during the crop growth stage of three cultivars of Lupinus albus (white lupin) under field conditions, taking into account sowing time effects (autumn and early spring) and cultivar effects. Three sampling dates (from late vegetative to late grain growth stages) were evaluated. Seven isoflavones and four flavonoids were identified by LC-ESI-MS analysis. The isoflavone content was higher in leaves than in stems, and it was highest before flowering, whereas it decreased during maturity. Autumn-sown plants showed higher isoflavone content than early spring-sown plants, especially in late vegetative and early reproductive stages. Genistein 7- O-glucoside was the main isoflavone of leaves and stems in the late vegetative stages of early spring sowing, whereas genistein was the main isoflavone under autumn sowing. Variation among cultivars affected only marginally the total isoflavone content. No isoflavones were detected in seeds.