Quantitative sandwich ELISA for the determination of lupine (Lupinus spp.) in foods

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.     

Carbon and nitrogen mineralisation from green manures as alternative nitrogen sources in Mediterranean farming

The soil incorporation of green manures is a practice that can be used in sustainable agriculture and in organic farming, where nitrogen (N) sources are limited. The aim of this study was to evaluate balansa clover (Trifolium michelianum Savi), yellow lupine (Lupinus luteus L.) and ryegrass (Lolium multiflorum Lam.) as potential alternative N sources. A total of nine treatments were considered in this study: control, aerial of balansa clover, roots of balansa clover, aerial of yellow lupine, roots of yellow lupine, aerial of ryegrass, roots of ryegrass, mixture aerial + roots of yellow lupine and mixture aerial + roots of ryegrass. A laboratory incubation experiment was conducted under controlled conditions during 196 days and carbon and N mineralisation were followed. Results showed that green manures are appropriate N sources for Mediterranean farming. No significant differences in terms of N mineralisation were observed between aerial or roots biomass of the green manures. Besides, 37–55% of total N applied was mineralised in treatments amended with balansa clover or yellow lupine, whereas 13–21% of total N applied was mineralised in ryegrass. It can be concluded that the most efficient green manure for supplying mineral N to the succeeding crop was yellow lupine.     

Establishment of selenium uptake and species distribution in lupine, Indian mustard, and sunflower plants

Selenium has been recognized as essential for all mammals; therefore, its concentration level and speciation are of great concern. Plants are one of the main sources of selenium in the diet. Thus, inorganic selenium uptake and its transformation in different species were evaluated in Indian mustard (Brassica juncea), sunflower (Helianthus annus), and white lupine (Lupinus albus). More than 1.2 g x kg(-)(1) (dry matter) of Se was found in the aerial part of Indian mustard when growing on 1 mg x L(-)(1) of Se as Na(2)SeO(4), and approximately half this amount was determined in the leaves of the lupine, which is still quite high. Selenomethionine was the main selenium-containing amino acid identified in most of the extracts by HPLC-ICP-MS. The higher values were 6.8 and 14.5 mg x kg(-)(1) (expressed as Se in dry matter) in the leaves of lupine and sunflower, respectively. This is of great importance because some authors have considered the combination of this enriched material with non-enriched food as a source of selenium supplementation.     

Inbreeding and small population size reduce seed set in a threatened and fragmented plant species, Lupinus sulphureus ssp. kincaidii (Fabaceae)

Willamette Valley upland prairie in western Oregon, USA, has been reduced to less than 1% of its original historic range following European settlement in the 1850s. Lupinus sulphureus ssp. kincaidii (Kincaid's lupine), a threatened species and the primary larval host plant of the endangered Icaricia icarioides fenderi (Fender's blue butterfly), was historically a panmictic metapopulation. Habitat fragmentation may be causing many of the Kincaid's lupine colonies to display typical symptoms of inbreeding depression, such as low seed production. Hand outcrosses on bagged inflorescences significantly increased seed set and seed fitness compared to open pollination and within-colony pollen treatments. Natural seed set was positively correlated with an increase in the number of Kincaid's lupine patches, suggesting that population size limits seed set. An increase in fruit set was positively correlated with Kincaid's lupine raceme number, raceme density, and the number of lupine patches, demonstrating that floral display and population size increase pollinator service. Restoration of Kincaid's lupine populations should consider measures that lessen the effects of inbreeding depression, especially in small, isolated populations, for the long-term persistence of the species.     

Phosphorus nutrition of white lupine grown on a Calciaquoll

Abstract

White lupine (Lupinus albus L.) and navy bean (Phaseolus vulgaris L.) are legumes with and without the proteoid root characteristic, respectively. The influence of P (0, 40, 80, and 120 mg/kg of NaH₂PO₄‐P) and source of nitrogen (N) [inoculation with Bradyrhizobium lupini or Rhizobium phaseoli vs. NH₄NO₃] on phosphorus (P) uptake by navy bean and white lupine grown on a Calciaquoll low in available P and N was studied under greenhouse conditions. Both species responded to P fertilizer, but P accumulation in navy bean tops was much greater than in white lupine. Inoculated white lupine plants accumulated more P than their NH₄NO₃‐treated counterparts. Growth and P uptake of 17‐day‐old flax (Linum usitatissium L.) plants grown in pots previously planted to white lupine, but not in their navy bean counterparts, was severely retarded. White lupine did not enhance the availability of soil P in the calcareous soil.     

Flora and lepidoptera fauna adversely affected by invasive Lupinus polyphyllus along road verges

An increasing number of invasive species are changing ecosystems around the world. Road verges have commonly become the first footholds of non-native species in the new environments. Regularly mown road verges also offer habitats for meadow flora and fauna, which in Europe have suffered from the radical decline of semi-natural biotopes due to the agricultural modernization. We studied impacts of an invasive plant Lupinus polyphyllus on the plant and Lepidoptera species composition along road verges. The plant species composition was studied on 15 sites (with 1 m² quadrats) and butterflies and diurnal moths along 15 transects (with weekly censuses) in SE Finland, each site and transect representing equally lupine invaded verge and an adjacent non-lupine verge. The species richness and diversity of flora and the cover and species richness of low growing (<20 cm) species, in particular, was lower in lupine verges compared to non-lupine verges. Also, the abundance of butterflies was lower in lupine verges. As the lupine cover approached 100%, fewer butterflies were observed in lupine transects compared to the adjacent non-lupine transects and a higher proportion of individuals were flying. Our results suggest that the changes in plant species assemblages and lower plant species richness in lupine invaded areas had “bottom-up” effects on higher trophic levels. Further studies on the control of lupine are urgently needed, but meanwhile we suggest regular mowing before the lupines have shed their seeds, together with the removal of the cuttings, to be the best management option.     

Development of a real-time PCR for the detection of lupine DNA (lupinus species) in foods

Lupine flour, protein, and fiber have become common ingredients in food products. The association of lupine-related allergic incidents with peanut allergy is a cause for concern as the latter may bring about severe reactions. In this study, a hybridization probe-based real-time PCR assay for the detection of lupine DNA in foods was developed. Particular attention was paid to the specificity of the method, which was verified by analysis of DNA extracts from more than 50 potential food ingredients such as legumes, cereals, seeds, nuts, spices, fruits, and meat. The limit of detection of the method was determined as 0.1 mg/kg. The successful detection of the presence/absence of lupine DNA in 20 samples proved the suitability of the assay for the analysis of frequently encountered food matrices.     

Lupine induced "crooked calf disease" in Washington and Oregon: identification of the alkaloid profiles in Lupinus sulfureus, Lupinus leucophyllus, and Lupinus sericeus

Several lupines (Lupinus spp.) present on western U.S. rangelands contain alkaloids that are teratogenic to livestock and cause congenital birth defects in calves (crooked calf disease). Periodically, large losses of calves due to lupine-induced "crooked calf disease" occur in northern Oregon and eastern Washington state. Five lupine populations from this area representing three species (L. leucophyllus, L. sulfureus, and L. sericeus) were evaluated taxonomically and by gas chromatography/mass spectrometry, and the major alkaloids in each lupine species were identified. The teratogenic alkaloid anagyrine was present in both of the lupine species responsible for the high outbreaks in east-central Washington and northeastern Oregon. However, the alkaloid profiles of the two lupines identified as L. leucophyllus were dissimilar, as were the alkaloid profiles of the two lupines identified as L. sulfureus. Botanical classification is not sufficient to determine potential teratogenicity, and it must be followed by chemical characterization to determine risk to livestock.     

High-performance capillary electrophoresis with indirect UV detection for determination of alpha-galactosides in Leguminosae and Brassicaceae

A rapid, easy, and reproducible capillary electrophoresis method for determination of raffinose family oligosaccharides (alpha-galactosides) was developed. Sucrose, raffinose, stachyose, verbascose, and ajugose were determined with indirect UV detection at moderate alkaline pH 9.2, using pyridine-2,6-dicarboxylic acid as background electrolyte in a sodium tetraborate buffer with added cetyltrimethylammonium bromide. The separation efficiency measured by the number of theoretical plates (N) ranged from 1.4 x 10(5) to 2.3 x 10(5). The precision of the method, measured by the relative standard deviation (RSD), was less than 0.53% for the migration times and better than 3.4% for normalized areas (NA), considering all sugars except verbascose (RSD(NA) = 11.8%). Detection limits were about 110 microg/mL, corresponding to 150-320 microM. Relative response factors (RRF) were calculated on the basis of linearity studies and used for quantification of alpha-galactosides in a lupine sample (Lupinus angustifolius).     

Characterization of developing microbial communities in Mount St. Helens pyroclastic substrate

The 1980 eruption of Mount St. Helens created a unique opportunity to study microbial communities in a developing soil ecosystem containing little total carbon (C) or total nitrogen (N). We collected surface samples (0-5 cm) from areas near Mount St. Helens National Volcanic Monument 17 years after the eruption. The samples were from bare soil with no plant development, soil under living prairie lupine (Lupinus lepidus) and dead prairie lupine in the pyroclastic plain near Spirit Lake, Washington. We also collected soil from a nearby forested area. Phospholipid fatty acids (PLFAs) from pyroclastic materials were analyzed to determine changes in soil microbial composition. Total bacterial DNA was also extracted from the soils and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes and DNA sequence analysis of cloned 16S rRNA gene libraries were used to determine the influence of plants on microbial development. Both principal components analysis (PCA) of PLFA fingerprints and non-metric multidimensional scaling (NMS) of DGGE fingerprints distinguished the four soils. Lupine plants influenced the PLFA and DGGE fingerprints depending on the distance of the samples from the plant. DGGE and PLFA profiles from the forest soil were significantly different (P=0.001, based on Monte Carlo permutation test) from those of the bare soil and soil with live lupine. Bacterial clone libraries were constructed, and 800 clones were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) and grouped into operational taxonomic units (OTUs). A total of 51, 77, 58, and 42 different OTUs were obtained from forest soil, soil with live and dead lupine, and bare soil, respectively. Phylogenetic analysis revealed that 62% of the 228 OTUs were classified as Proteobacteria, Actinobacteria, Acidobacterium, Verrucomicrobia, Bacteroides, Cyanobacteria, Planctomycetes, and candidate divisions TM7 and OP10. Members of Proteobacteria represented 29% of the OTUs. Thirty-eight percent of the OTUs could not be classified into known bacterial divisions. This study emphasized the role of prairie lupine in the establishment of pioneering microbial communities and the subsequent roles the biotic components played in improving the quality of pyroclastic soil.     

Marzipan: polymerase chain reaction-driven methods for authenticity control

According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected.     

The Effect of Legume Species Grown as Cover Crops in Olive Orchards on Soil Phosphorus Bioavailability

Some legume species are able to utilize phosphorus (P) from sparingly soluble P sources benefiting companion crops or those following in the rotation. Lupinus albus, Vicia villosa, and a mixture of eleven annual pasture legumes were used in olive orchards as mulched cover crops as a means of increasing the soil P availability. By soil testing and growing bioindicator P plants in the next season, it was possible to detect a slight but consistent increase in soil P availability. The results indicated that the increase in soil P availability was mainly due to the mineralization of the high P content legume residues, rather than the direct effect in the rhizosphere of the living plants. The results also suggested that the good adaptation of white lupine to low P environments might be due to a high internal P use efficiency of this species, producing high dry matter yields with low P concentration in their tissues.     

Resource availability, matrix quality, microclimate, and spatial pattern as predictors of patch use by the Karner blue butterfly

Determination of which aspects of habitat quality and habitat spatial arrangement best account for variation in a species’ distribution can guide management for organisms such as the Karner blue butterfly (Lycaeides melissa samuelis), a federally endangered subspecies inhabiting savannas of Midwest and Eastern United States. We examined the extent to which three sets of predictors, (1) larval host plant (Lupinus perennis, wild lupine) availability, (2) characteristics of the matrix surrounding host plant patches, and (3) factors affecting a patch’s thermal environment, accounted for variation in lupine patch use by Karner blues at Indiana Dunes National Lakeshore, Indiana and Fort McCoy, Wisconsin, USA. Each predictor set accounted for 7-13% of variation in patch occupancy by Karner blues at both sites and in larval feeding activity among patches at Indiana Dunes. Patch area, an indicator of host plant availability, was an exception, accounting for 30% of variation in patch occupancy at Indiana Dunes. Spatially structured patterns of patch use across the landscape accounted for 9-16% of variation in patch use and explained more variation in larval feeding activity than did spatial autocorrelation between neighboring patches. Because of this broader spatial trend across sites, a given management action may be more effective in promoting patch use in some portions of the landscape than in others. Spatial trend, resource availability, matrix quality, and microclimate, in general, accounted for similar amounts of variation in patch use and each should be incorporated into habitat management planning for the Karner blue butterfly.     

Phosphorus uptake from struvite is modulated by the nitrogen form applied

Next to nitrogen, phosphorus (P) is the most limiting nutrient for plant production worldwide. To secure food production, new nutrient management strategies using alternative P sources instead of mined P fertilizers need to be implemented. Struvite (MgNH₄PO₄ · 6 H₂O) is a promising example of a recycled mineral P fertilizer. Besides positive agronomic results regarding crop yields, further investigations are required to improve the use efficiency of the product and thereby increase its value. Using an automated plant phenotyping platform, we investigated the dynamic response to struvite by two plant species (lupine and maize) with diverse P acquisition strategies in an acidic sandy substrate. Although at three weeks after germination both maize and lupine had reduced leaf area in the struvite treatments compared to the commercial triple superphosphate (TSP), from week four onwards struvite plants grew larger than the TSP‐treated plants, indicating a slow release fertilizing effect. Greater P uptake efficiency (g / root length), but reduced root length were observed in the combined treatment of struvite and ammonium, in comparison to struvite and nitrate. We propose that rhizosphere acidification in response to ammonium uptake may enhance P recovery from struvite. A possible additional acidification effect by lupine root exudation might explain the higher P uptake efficiency in this species compared to maize. We conclude that struvite combined with ammonium can be used as a sustainable slow‐release P fertilizer on acidic sandy soils.     

Interactions between crop residues application and mycorrhizal developments and some soil-root interface properties and mineral acquisition by plants in an acidic soil

The addition of plant residues and the appropriate management of arbuscular mycorrhizal (AM) symbioses have been tested in an acidic soil, an Andisol from Southern Chile, to ascertain whether these agro-technologies help plants to withstand potential mineral deficiency and the toxicities inherent to the low pH conditions. Firstly, the effects of legume (lupine) and non-legume (wheat) crop residues on some key root-soil interface activities (including AM development), on mineral acquisition by the plants, and on the yield of wheat growing in the test Andisol were investigated in a pot experiment under greenhouse conditions. Both lupine and wheat residues were added at a rate equivalent to 300 g m^-2 to the natural soil. These organic amendments increased soil pH (wheat more than lupine), P availability and AM development (lupine more than wheat), plant performance and mineral acquisition (wheat more than lupine). Because of an increase in mycorrhizal activity, which appeared to be involved in the effect of the added crop (particularly lupine) residues, the role of the AM symbiosis was further investigated in a tailored inoculation assay, using a selected AM fungus (Glomus etunicatum), in interaction with lupine and wheat residues. A significant effect of AM inoculation on the reduction of Zn and Cu, and Mn and Al acquisition was demonstrated, which could be of interest in acid soils with regard to potential toxicity problems.     

The role of alkaloids in the feeding behaviour of slugs (Gastropoda: Stylommatophora) as pests of narrow-leafed lupin plants

The degree of damage done to lupin plants by slugs is highly dependent on the species and the cultivar of plant. A study was carried out, in laboratory conditions and on small plots, to investigate the extent of damage done to plants of eight cultivars of narrow-leafed lupin when exposed to grazing by Arion vulgaris, Arion rufus and Deroceras reticulatum. Five sweet and three bitter cultivars of lupin were selected, respectively, having low- and high-alkaloid content. The contents of the dominant lupine alkaloids in these plants were determined. It was found that the susceptibility of the plants to slug damage is dependent on their content of alkaloids. The plants of high-alkaloid cultivars (Karo, Mirela and Oskar) were less heavily damaged by the studied species of slugs. The greatest differences were recorded for A. vulgaris and D. reticulatum, while the differences for A. rufus were smaller. For all studied slug species, the differences were statistically significant. The results of carried out experiments may potentially be of use in the selection of cultivars of Lupinus angustifolius to be grown in areas inhabited by these slug species.     

Different leguminous pre-crops increased yield of succeeding cereals in two consecutive years

Leguminous pre-crops are an important source of green manure in organic crop rotations for improving soil fertility and achieving high yields of cereals. We aimed to study the potential of various leguminous species, other than the traditionally cultivated red clover (Trifolium pratense L.), as green manure pre-crops for subsequent cereals. The use of different legume species enables to exploit advantages of specific legumes in organic cereal production. In order to test the legumes as pre-crops for cereals, we carried out trials located in the temperate climate zone of northeast Europe (58°44′59.41″ N, 26°24′54.02″ E). We sowed the following perennial legumes as pre-crops: red clover, alsike clover (Trifolium hybridum L.) and Washington lupine (Lupinus polyphyllus Lindl.), biennial white sweet clover (Melilotus albus Medik.) and annual Alexandria clover (Trifolium alexandrinum L.), and crimson clover (Trifolium incarnatum L.). Timothy (Phleum pratense L.) was used as a control. The leguminous pre-crops were followed by three spring cereals (barley, oat and spring wheat) and two winter cereals (rye and winter wheat). We tested the first-year after-effect (all cereals) and second-year after-effect (only barley and oat) of pre-crops on the grain yield of cereals. Perennial and biennial legume species produced the highest dry matter yield and contained the highest amount of nutrients, especially nitrogen, compared to annual species. All subsequent cereals produced significant extra yields after each leguminous pre-crop in the following two years, although the effect was smaller in the second year. The most suitable pre-crops for spring cereals were red and alsike clover followed by lupine, whereas the best pre-crops for winter cereals were sweet clover and annual clovers. Our results show the potential of various leguminous pre-crop species as valuable sources of green manure in organic crop rotation.     

Impact of precursor crop and nitrogen fertilizer on the productivity of finger millet based cropping system and soil fertility in Lake Tana basin of Ethiopia

Abstract

Traditional short-term diversification of annual crops for the purpose of improving soil fertility and food security is a common practice in Ethiopia. However, the best cropping systems have not been selected yet though field research. Experiments were conducted for two years with an objective of establishing sustained finger millet based short term cropping sequence in two major finger millet producing areas of Ethiopia. The experiments were laid out in a split-plot arrangement with six precursor crops (haricot bean, maize, sweet lupine, niger seed, wheat, and teff) as a main plot factor, where determined residues were retained in the main plot after harvesting and two nitrogen fertilizer levels (30 and 60?kg ha^?1) applied for finger millet as sub-plots with three replications. Soil analysis result examined immediately after finger millet harvest showed a significant effect of precursor crops and nitrogen fertilizer rates on soil properties. Planting finger millet following lupine increased the soil organic carbon, total nitrogen and cation exchange capacity by 17.37%, 65.50%, 26.15%, respectively, over planting finger millet following teff. Use of different amount of nitrogen fertilizer likely contributed to observed differences in residual soil nutrients. Accordingly, finger millet following lupine with the addition of 60?kg ha^?1 nitrogen fertilizer gave significantly higher finger millet grain yield (3.24 tone ha^?1) and relative production (22.37%) and economic (40.7%) efficiency. This system with their high sustainability and multiple benefits can be readily accepted by the farming community.     

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.     

A novel approach for the detection of potentially hazardous pepsin stable hazelnut proteins as contaminants in chocolate-based food

Contamination of food products with pepsin resistant allergens is generally believed to be a serious threat to patients with severe food allergy. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to measure pepsin resistant hazelnut protein in food products. Capturing and detecting rabbit antibodies were raised against pepsin-digested hazelnut and untreated hazelnut protein, respectively. The assay showed a detection limit of 0.7 ng/mL hazelnut protein or <1 microg hazelnut in 1 g food matrix and a maximum of 0.034% cross-reactivity (peanut). Chocolate samples spiked with 0.5-100 microg hazelnut/g chocolate showed a mean recovery of 97.3%. In 9/12 food products labeled "may contain nuts", hazelnut was detected between 1.2 and 417 microg hazelnut/g food. It can be concluded that the application of antibodies directed to pepsin-digested food extracts in ELISA can facilitate specific detection of stable proteins that have the highest potential of inducing severe food anaphylaxis.