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.

Effect of genotype and environment on fatty acid composition of Lupinus albus L. seed.

Six cultivars of Lupinus albus L. (white lupin) were grown in two subcontinental-climate environments and one Mediterranean-climate environment in Italy, to assess the influence of genotypic (G) and genotype×environment (GE) interaction effects on grain yield and grain content of oil, total saturated fatty acids (FAs), polyunsaturated FAs, monounsaturated FAs, and ω-3/ω-6 FA ratio. The variance of genotypic effects was much larger than the GE interaction variance for all variables, except for grain yield, indicating that oil content and FA composition of different varieties can be assessed reliably in just a few test environments. Gas-chromatographic analyses highlighted that linoleic acid and α-linolenic acid were in the range 1.76-4.76mg/g flour (7.79-15.81% of total FAs) and 1.17-3.14mg/g flour (5.40-10.36% of total FAs), respectively. As a consequence, the analysed lupin seeds exhibited a very favourable ω-3/ω-6 FA ratio, ranging from 0.49 to 0.79.

Parameters for the evaluation of the thermal damage and nutraceutical potential of lupin-based ingredients and food products.

Foods based on sweet lupin proteins are gaining attention from industry and consumers because of their possible role in the prevention of cardiovascular disease. When promoting lupin-based foods for inclusion in a daily diet, the thermal damage suffered during processing is of relevance to the bioactive and nutritional quality of the food product. N-(2-furoylmethyl)-L-lysine (furosine) quantification demonstrates that currently available sweet lupin protein isolates have a thermal damage comparable to or lower than other traditional food ingredients, and are a good source of lysine in non-dairy products. In lupin-based foods claiming to have cholesterol-lowering potential, shotgun proteomics offers itself as a fast and effective screening method for assessing the biological availability of active peptides. Such a method is readily applicable to other legume-enriched food products.

Hydrolytic degradation of azimsulfuron, a sulfonylurea herbicide.

The chemical degradation of the herbicide azimsulfuron was investigated in aqueous solutions at different pH values. The hydrolysis rate, determined by HPLC analyses, was pH dependent and was much faster in acidic than in neutral or weakly basic conditions. The metabolites formed at different pH values were compared with standards when possible or isolated and identified using ESI-LC-MS/MS, (1)H NMR and (13)C NMR. The two main products of hydrolysis in mild acidic solution were identified as 2-amino-4,6-dimethoxy-pyrimidine and 2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide, both produced as a result of the sulfonylurea bridge cleavage. Under basic conditions, a new product, a substituted 2-pyrimidinamine, deriving from the contraction of the sulfonylurea bridge, was isolated and completely characterized for the first time.

Optimization of a pilot-scale process for producing lupin protein isolates with valuable technological properties and minimum thermal damage.

This paper describes a pilot process for obtaining protein isolates from white lupin seed with improved water solubility and technofunctional properties as well as reduced thermal damage. After a careful optimization of the process parameters, two valuable food ingredients were prepared: lupin protein isolate type E, with a useful emulsifying capacity, and lupin protein isolate type F, with a high capability of foam formation and stabilization. The spray-drying process was particularly critical for inducing some thermal damage, but a careful selection of the conditions permitted ingredients having only marginally impaired lysine bioavailability to be obtained. The reproducibility of the protein extraction process was tested on two different lupin varieties.

Investigations on the high molecular weight foaming fractions of espresso coffee.

The target of the present work was the chemical, technological, and sensorial characterization of the brown polymers (foaming fractions) of freshly prepared espresso coffee. The total foaming fraction (TFF) was precipitated with ammonium sulfate from the defatted freshly prepared beverage and then subfractionated by adding 2-propanol/water to give an insoluble fraction (foaming fraction A, FFA) and a soluble fraction (foaming fraction B, FFB). The former is almost colorless, has a higher molecular weight and a lower nitrogen content, and contains mostly polysaccharides, whereas the latter has a lower molecular weight and a higher protein/melanoidin content, which results in a darker color. FFB showed greater foaming capability, but FFA contributed to the stability of the foam. FFB was further fractionated with solid-phase extraction and characterized by different analytical methods (size exclusion chromatography, UV, HPLC-DAD, 1H NMR). All of the melanoidin-rich fractions showed antioxidant properties with the 2,2-diphenyl-1-picrylhydrazyl hydrate method.

Biodegradation of chlorsulfuron and metsulfuron-methyl by Aspergillus niger.

In this work, investigations were performed under laboratory conditions of the degradation ability by a common soil fungus, Aspergillus niger, toward chlorsulfuron and metsulfuron-methyl. The results were very encouraging (79% for chlorsulfuron and 61% for metsulfuron-methyl of total degradation), especially compared to those registered in our previous studies with a Pseudomonas fluorescens strain B2 (about 21 to 32%). Furthermore, the chemical degradation of the two compounds was studied and two products (1[2-methoxy-benzene-1-sulfonyl]-7-acetyltriuret and 1[2-chlorobenzene-1-sulfonyl]-7-acetyltriuret) were isolated and characterised by hydrolysis in acidic conditions. Our aim in the future will be the identification of intermediate metabolites by HPLC and LC-MS analyses in order to identify the degradative pathway by the fungal strain and to compare this to those obtained by chemical degradation and by P. fluorescens strain.

Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods.

Lupin proteins are gaining attention to replace animal proteins and other plants ingredients in several foods such as bakery products, imitation dairy and meat products, and beverages. One of the major safety issues of lupin-based foods is the presence of quinolizidine alkaloids (QAs), bitter compounds produced by lupin plants as a defense mechanism against predators. In mammals, QA intoxication is characterized by trembling, shaking, excitation, and convulsion. Lupanine and sparteine, the most common QAs, show acute oral toxicity due to neurological effects leading to the loss of motor co-ordination and muscular control. In this paper, 27 samples of lupin-based products, i. e., flours, protein isolates, and food (either model or commercially available ones), were analyzed for evaluating the QA content using a method based on GC/MS. All the analyzed samples were safe since they respect the maximum limit of 200 mg/kg fixed by the Health Authorities of Australia, New Zealand, Great Britain, and France, that have regulated this topic. The QA contents were particularly low in protein isolates and in foods containing these ingredients, indicating that their use is a very effective tool for keeping low the daily intake of QAs.

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.

Characterization of field-isolates and derived DMI-resistant strains of Cercospora beticola.

Cercospora beticola strains with laboratory induced resistance to tetraconazole were compared with their parental WT sensitive strains to evaluate the effects of resistance on fitness and assess whether any change in the sterol biosynthetic pathway was associated to the reduced fungicide sensitivity. In vitro growth rate on agar media and pathogenicity were found to be negatively affected by resistance. The main functional sterols in C. beticola WT strains under investigation were ergosterol, brassicasterol and ergosta-7,22-dienol. Resistant strains showed the same qualitative sterol composition, ruling it out as, per se, a cause for resistance. On the basis of the sterols detected both in sensitive and resistant strains, a possible biosynthetic pathway to the three functional sterols is proposed. Tetraconazole treatment caused, in all sensitive strains, the immediate accumulation of 14alpha-methylated sterols, which, for inhibitor concentrations up to EC50 values, were, in order of abundance, 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha-diol, eburicol and obtusifoliol. However the data do not support a critical role of the 14-methyl-3,6-diol derivative in the growth arrest of C. beticola. As main difference between sensitive and resistant strains, the formers were found to accumulate higher amounts of 14alpha-methylated sterols. Although the data do not allow to establish a specific mechanism for resistance, some molecular mechanisms such as target site alterations and sterol biosynthetic pathway can be ruled out as a possible cause for reduced sensitivity.

Biodegradation of chlorsulfuron and metsulfuron-methyl by Aspergillus niger in laboratory conditions.

Two sulfonylurea herbicides, chlorsulfuron and metsulfuron-methyl, were studied under laboratory conditions, in order to elucidate the biodegradation pathway operated by Aspergillus niger, a common soil fungus, which is often involved in the degradation of xenobiotics. HPLC-UV was used to study the kinetic of degradation, whereas LC-MS was used to identify the metabolites structure. In order to avoid the chemical degradation induced by a decrease in pH, due to the production of citric acid by the fungus, the experiments were performed in a buffered neutral medium. No significant degradation for both compounds was observed in mineral medium with 0.2% sodium acetate. On the contrary, in a rich medium, after 28 days the degradations, chemical degradation excluded, were about 30% for chlorsulfuron and 33% for metsulfuron-methyl. The main microbial metabolites were obtained via cleavage of the sulfonylurea bridge. In addition the fungus seems to be able to hydroxylate the aromatic ring of chlorsulfuron. In the case of metsulfuron-methyl the only detected metabolite was the triazine derivative, while the aromatic portion was completely degraded. Finally, the demethylation of the methoxy group on the triazine ring, previously observed with a Pseudomonas fluorescens strain, was not observed with A. niger.

Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells.

White lupin (Lupinus albus, L.), a widely cultivated crop that has been consumed for many years in Western Europe, may provide a useful alternative for individuals wishing to substitute animal with plant proteins for cardiovascular disease prevention. Lupin seeds have a very low content of isoflavones, and lupin protein isolates are essentially isoflavone free. In rats fed a casein-based cholesterol + cholic acid diet, a relatively low daily intake (50 mg/d by gavage for 2 wk) of total lupin protein extract reduced plasma total and VLDL + LDL cholesterol concentrations by 21 and 30%, respectively (both P<0.001). In an attempt to elucidate the lipid-lowering mechanism, LDL receptor activity was evaluated in a human hepatoma cell line (HepG2). In this model, the lupin total protein extract was essentially inactive, whereas one purified minor protein component, conglutin gamma, had a remarkable upregulatory effect, with maximal increases of 53 and 21% (both P<0.05) for LDL uptake and degradation, respectively. This initial study indicates that lupin, although isoflavone free, has hypocholesterolemic activity similar to that of other leguminous proteins in an established animal model. Further, the cholesterol reduction appears to be associated with stimulation of LDL receptors by a well-defined protein component of the lupin seeds as demonstrated by in vitro studies.