Hydrolysis of the toxic constituents (vicine and convicine) in fababean (Vicia faba L.) food preparations following treatment with β-glucosidase

Several experiments were carried out to determine whether the concentrations of vicine and convicine in fababean food preparations were reduced following heat and β-glucosidase treatment. Heating fababeans to relatively high temperatures was not effective in reducing the concentration of these glycosides. Treatment of cooked fababeans (fababean paste) with ground raw almond powder, which is a rich source of β-glucosidase (EC 3.2.1.21) activity, resulted in the hydrolysis of vicine and convicine. The degree of hydrolysis ranged from partial to complete and was dependent on the duration of the treatment period (1 to 24 h), the temperature at which treatment was carried out (1 to 30°C), the pH (6.4 to 4.6) as adjusted by the addition of lemon juice and the amount of ground almonds added to the fababean paste. For example, 88 to 89% of the glycosides were hydrolysed when 1 g of fababean paste was mixed with 0.1 g of almond powder and 0.1 ml of lemon juice and incubated at 30°C for 3 h. This study demonstrated that the concentration of vicine and convicine and their corresponding aglycones can be greatly reduced in fababean food preparations by enzymic hydrolysis.     

Removal of favism-inducing factors vicine and convicine and the associated effects on the protein content and digestibility of fababeans (Vicia faba L)†

Ingestion of fababeans is associated with precipitation of the haemolytic disease favism in certain glucose-6-phosphate dehydrogenase-deficient humans. Hence their incorporation into weaning foods has not yet been practised. Few, if any, attempts to detoxify fababeans have so far been made. The present investigation has evaluated the effects of a series of treatments designed for vicine and convicine extraction and hydrolysis followed by oxidation of their pyrimidine moieties (divicine and isouramil). The effects of seed germination and oxidative treatment by hydrogen peroxide have also been examined. Extracts of treated samples were assayed for residual vicine and convicine. Toxicity was monitored by the changes in vitro of reduced glutathione (GSH) of red blood cells (RBCs) from Sprague Dawley rats deliberately made deficient in glutathione reductase activity to mimic favism-susceptible human RBCs. Treatments of whole cotyledons resulted in recovery of 59–93% vicine and 50–70% convicine originally present in the seeds. Treatments of fababean powders, however, were capable of lowering the vicine content by 94–100% and convicine content by 100%. Germinated seeds showed a drop in vicine content of 86% and their hydrogen peroxide treatment 91–93%. Convicine was totally absent in germinated and oxidised seeds. The results of toxicity assays were concomitant with vicine and convicine analyses. Protein content of original fababean powders was well recovered (up to 94.00%) and its digestibility was almost complete (99.34%). © 1999 Society of Chemical Industry     

Effect of genotype and environment on vicine and convicine levels in fababeans (Vicia faba minor)

The concentrations of vicine and convicine (1.94 and 0.83%, dry basis, respectively) in fababeans were highest in young seeds and decreased rapidly with maturity. By 21-28 August 1978 the concentrations had levelled off and the rank order of four cultivars, which differed widely in days required to mature, remained unchanged for the rest of the growing season. Analysis of variance on 36 fababean cultivars grown at different locations and years in Saskatchewan showed that genotype and environment had highly significant (P=0.01) effects on the glucoside level of fababean samples. Although lower in concentration (mean=0.31%), convicine showed greater variability among cultivars than vicine (mean = 0.66%). The glucoside contents of 242 diverse cultivars grown at Saskatoon ranged from 0.44 to 0.82% for vicine and 0.13 to 0.64% for convicine with the lowest cultivar containing 0.62% total glucoside. Individual seeds of a cultivar differed greatly in glucoside content and could become another source of variability for plant breeding and selection of low-glucoside cultivars. Hulls were relatively free of glucosides but, despite variations in hull content, it was unnecessary to dehull samples before analysis.     

Development of a simple procedure for the complete extraction of vicine and convicine from fababeans (Vicia faba? L.)

Egyptian foods prepared from fababeans contain variable and relatively high concentrations of vicine. Vicine and convicine can be readily extracted from whole or dehulled fababeans. The rate and completeness of extraction depends upon size of beans, the presence or absence of testa, the pH, temperature and volume of the aqueous extracting solution, the duration of the extraction procedure and the frequency with which the extracting solution is changed. In a typical experiment vicine and convicine were almost completely removed from whole or dehulled fababeans when they were extracted at 20 or 40°C with 1% glacial acetic acid (0.17M) or water over a 72 h period with changes in the extracting media every 24 h. This appears to be the most practical procedure for extraction. The low pH of the acid solution prevents bacterial fermentation that occurs when water is used. Water at temperatures greater than 70°C is a highly effective extracting agent. Extraction of fababeans with acetic acid does not markedly affect the concentration of protein (N × 5.7), fat or fibre. Ash content, however, was reduced by a factor of 2 and the total dry matter content of the extracted beans was reduced by 16–17% in whole beans and 20–21% in dehulled beans.     

Turnover and hydrolysis of vicine and convicine in avian tissues and digesta

The objectives of this study were to establish factors influencing the absorption, excretion and hydrolysis of vicine and convicine in chicks. Blood vicine, following the oral administration of a single dosage of vicine into the crop of young chicks, reached maximum concentrations within 3 h. It was nearly completely removed from the blood within 12 h and had a half-life of approximately 4.5 h. The accumulation and excretion patterns of vicine in the liver and kidney were similar to those of the blood except that the concentrations were much higher in these tissues, particularly the kidney. Bile also contained a very high concentration of vicine which tended to accumulate following the decline in other tissues. These results together with the appearance of vicine in the urine of colostomised birds suggest that vicine is excreted in the urine and bile. Convicine in contrast to vicine was not absorbed by the chick. In-vitro studies were carried out with tissue and digesta homogenates from the chick in order to establish the site(s) at which vicine and convicine were hydrolysed to their aglycone forms. The results demonstrated that neither vicine nor convicine were hydrolysed in the presence of liver, kidney, intestinal wall or caecal wall homogenates, digesta from the large intestine or by enzymes present in whole or ground fababeans. They were, however, slowly hydrolysed in the presence of 0.1N HCl at 37°C and very rapidly hydrolysed by digesta from the caeca. Antibiotic additions to the diets markedly reduced the in-vitro rate of hydrolysis of these compounds. The latter results suggest that vicine and convicine are hydrolysed by microorganisms in the caeca of the chick but are not hydrolysed by the micro-organisms in the gastrointestinal tract, by endogenous tissue enzymes or by enzymes present in fababeans and only minimally hydrolysed by the low pH of the stomach.     

Purification,identification and quantification of an egg-weight-depressing factor (vicine) in fababeans (Vicia faba L.)

An egg-weight-depressing factor in fababeans was isolated in pure form from a white precipitate which had been shown in a previous study, to contain a concentrated form of this factor. The analytically pure compound was obtained in colourless, needle crystals. Its melting point was 242-244°C, it contained 17.4% nitrogen but did not react with ninhydrin. The spectra had adsorption maxima at 274, 276 and 269 nm in acidic, neutral, and alkaline solutions, respectively. These and other properties were identical to those of vicine [2,6-diamino-4,5-dihydroxy pyrimidine,5-(β-D-glucopyrano-side)] a compound known to be present in fababeans. Vicine and convicine [2,4,5-trihydroxy, 6-aminopyrimidine,5-(β-D-glucopyranoside)]; compounds having similar chemical and probably physiological activity, were resolved on a cation-exchange column and were detected by monitoring at 280 nm. Quantitative results obtained with the procedure was shown to be similar to those obtained with two other procedures. This method, however, was superior to other methods as it gave individual values for both vicine and convicine rather than a composite value; sample preparation was simple and the method could readily be adapted to automatic analysis.     

A modified procedure for the purification of vicine and convicine from fababeans (Vicia faba L.)

A procedure was developed for the simultaneous isolation of pure vicine [2,6-diamino-4,5-dihydroxypyrimidine 5-(β-D glucopyranoside)] and convicine [2,4,5-trihydroxy-6-aminopyrimidine 5-(β-D glucopyranoside)] from dehulled fababeans. The procedure involved the extraction of the glycosides with an ethanol-water (70:30, by vol.) solution, concentration of the extract to a minimal volume and adjustment of the pH of the aqueous extract to 7.2. Mixed vicine and convicine crystals, which were formed over a period of from 1 to 4 days, were harvested by centrifuging and washed with water and ethanol. Vicine was readily obtained from the mixed crystals by extraction at a low pH and recrystallised by adjustment of the pH to neutrality. Convicine, which is relatively insoluble in an acidic medium, was solubilised in an alkaline solution and recrystallised at a neutral pH.     

Metabolism of vicine and convicine in rat tissues: absorption and excretion patterns and sites of hydrolysis

The objectives of this study were to determine whether dietary vicine and convicine were absorbed by rat tissue, and to determine their excretion patterns and/or sites of degradation. Orally administered vicine and convicine were excreted in relatively low amounts via the kidney and faeces. However, no vicine or convicine was detected in the blood, liver, kidney or muscle tissue of rats which had been fed these compounds. In-vitro studies demonstrated that vicine and convicine were not hydrolysed in liver, kidney, muscle, caecal wall or intestinal wall homogenates. In contrast, digesta samples from the large intestine and caeca were able to rapidly hydrolyse these compounds, with the concomitant formation of new compounds. Digesta from the stomach and small intestine promoted the slow hydrolysis of these compounds, as did fresh faecal samples. These results would suggest that vicine and convicine are absorbed by the rat in only limited amounts, are not hydrolysed by rat tissues, and are rapidly cleared from tissue via the kidney. The bulk of the dietary vicine and convicine are hydrolysed in the large intestine and caecum.     

The Distribution of Pyrimidinone Glucosides in Developing Seedlings of Vicia faba and Vicia narbonensis

The distribution of the pyrimidinone glucopyranosides vicine and convicine was investigated in resting seed and axenic developing seedlings up to 17 days of age in Vicia faba cv Troy, a low seed pyrimidinone glucopyranoside mutant (MTG5) derived from Troy, and a related species V narbonensis. The lower amounts of total vicine and convicine in resting seed of MTG5 were due primarily to a reduction in cotyledon pyrimidinone glucopyranosides, with the amount in the embryo axis much less affected. Distribution within developing seedlings revealed consistent patterns. A pulse of vicine appeared in both species in stems and roots at day 4 to day 7, then declined. Amounts of convicine rose initially then declined in seedling stems of both species, whereas in roots the rise in convicine continued throughout the period. The rise in convicine in the roots of V narbonensis could not have been accounted for by conversion from reserves of vicine. The low pyrimidinone glucopyranoside mutant MTG5 had less vicine initially in stems and roots than both Troy and V. narbonensis but by 17 days this had risen close to the levels of Troy and exceeded those of V narbonensis. By flowering, amounts of vicine and convicine in glass-house grown plants were similar in MTG5 and Troy. The levels of both compounds in the roots of MTG5 exceeded those of the sown seed. Consequently, both V faba and V narbonensis have the capacity to synthesise vicine and convicine in young plants. A mutation in a gene controlling levels of pyrimidinone glucopyranosides in the developing seed of V faba does not suppress the biosynthetic ability of young plants.     

The concentration of vicine and convicine in Vicia faba and some related species and their distribution within mature seeds

Mature seeds from a range of species from the Vicieae tribe were analysed for vicine and convicine content. Quantifiable amounts of vicine were found in all the Vicia species analysed but neither glucopyranoside was detected in Pisum or Lathyrus. The concentrations of both vicine and convicine were similar for both Vicia faba and V bithynica but in all the other Vicia species glucopyranoside concentrations were significantly lower. Seeds from three faba bean lines and one each from V narbonensis and V bithynica were dissected into their constituent seed parts and these parts analysed. Only trace quantities were found in the seed coat but concentrations of vicine ranging from 39 g kg^?1 to 81 g kg^?1 were detected in the radicle samples. The implications of these results in relation to the role of these compounds in the plant are discussed.     

Effects of intraperitoneally injected vicine and convicine on the rat: Induction of favism-like signs

Rats were injected intraperitoneally with single or several successive doses of vicine or convicine. Single injections of these compounds at different concentrations caused increased respiration rates, generalised cyanosis, abdominal convolutions and, after several hours, death which appeared to be caused by asphyxiation. The tissues of the dead animals were engorged with dark brown blood and the large intestine and caecum contained entrapped gases and watery digesta and faecal matter. A second study demonstrated that injected vicine and convicine were cleared from the intraperitoneal cavity over several hours via the kidney and large intestine and that they were cleaved in the digesta of the large intestine and colon to divicine and isouramil. In a third study, rats were injected daily for 10 days with vicine or convicine. There were increases in spleen weight and blood monocytes and neutrophil counts and decreases in liver weight, blood glutathione and glucose concentrations, and lymphocyte counts. Blood from rats pretreated in vivo with convicine was shown to have an altered ultraviolet absorbance pattern. A similar pattern developed in vitro only in the presence of the aglycones of vicine (divicine) or convicine (isouramil) but not in the presence of the compounds themselves. The results of these studies demonstrate that vicine and convicine when injected intraperitoneally into the rat are converted to their aglycones which cause signs similar in many respects to those observed in the human metabolic disease, favism.     

Extraction of favism‐inducing agents from whole seeds of faba bean (Vicia faba L var major)

The objective of the present study was to establish a suitable procedure by which favism‐inducing glycosides vicine and convicine could be completely removed from whole seeds of faba beans without distorting their shape or reducing their nutritive value. Detoxification procedures carried out were extraction by water or, 10 g l^?1 acid solutions (acetic, citric and phosphoric acid) using stepwise or continuous soaking procedures. Three cultivars of the beans were used in the study. In the stepwise soaking procedures, the seeds were soaked in either water or acid solution at 40 °C for different periods while in the continuous flow techniques, the solutions were constantly passed through a seed reservoir under different time–temperature–flow rate conditions. Extraction of vicine and convicine increased with increasing temperature and period of soaking and/or continuous flow of soaking solutions. Neither stepwise soaking nor autoclaving could effectively remove the favism factors from the whole seeds. The best results were obtained with continuous flow soaking in tap water, giving vicine‐ and convicine‐free seeds. The protein contents of treated seeds were only slightly affected and their physical structure remained intact. Thus, it was concluded that the latter procedure was the method of choice for detoxification of dry whole seeds. Copyright © 2005 Society of Chemical Industry     

Content and distribution of vicine,convicine and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA during germination and seedling growth of two <Emphasis Type="Italic">Vicia faba</Emphasis> L. varieties

This work examined the content and distribution of the pyrimidine glucosides vicine and convicine, and the nonproteic amino acid l-DOPA in cotyledons and embryo axes along the germination and seedling growth of Vicia faba L. vars. Alameda and Brocal. The behaviour of these compounds was similar in both varieties. Vicine and convicine, implicated in favism disease, slowly declined in cotyledons. In embryo axis, vicine levels were sharply reduced and convicine amount was slightly increased as assay progressed. Total pyrimidine glucosides remained unchanged in the whole plant during the study. l-DOPA only appeared in the embryo axis of V. faba seeds and the highest content was observed in Brocal variety at 6 DAI (days after imbibition). The information provided in this study could be valuable for a possible role of embryo axis, rich in l-DOPA, for the treatment of Parkinson’s disease and also as a choice ingredient for functional foods or as nutraceutical.     

Nutritional and functional properties of Vicia faba protein isolates and related fractions

The goal of this research was the characterisation of Vicia faba (broadbean) protein isolates and related fractions in order to determine whether this grain legume could be used for production of high quality protein products and other fractions rich in functional components. Alkaline extraction of the defatted seed flour, followed by precipitation at the isoelectric pH, yielded a 92% protein isolate with a high oil absorption capacity. The contents of the favism-inducing glycosides, vicine and convicine, in the isolate were reduced by more than 99% as compared to the original flour, although the amino acid composition was similar to that of the flour. Some of the by-products of protein isolate production may also be of interest from a nutritional and functional point of view. Thus, the oil resulting from hexane extraction of the flour is rich in unsaturated fatty acids, and polyphenols (resulting from extraction of the defatted flour with acetone) showed a high ABTS radical-scavenging activity. In addition, the solid residue (resulting from protein solubilisation) was high in fibre and showed good water absorption. These results show good nutritional and functional properties in V. faba protein isolates and related fractions, which may favour the revalorisation of this traditional bean crop.     

The enzymic deacylation of p-nitrophenyl esters and phosphatidylcholine in vicia faba minor

Two enzymic deacylating activities have been detected in crude extracts of small fababeans—the hydrolysis of p-nitrophenyl fatty acyl esters and that of phosphatidylcholine. Kinetic studies demonstrated differences between the two activities with respect to substrate inhibition and to the effects of pH, Triton X-100, calcium chloride and mercuric chloride. The sequential activity of phospholipases A₁ and A₂, and lysophospholipase, in fababeans is postulated, and some similarity is indicated between the hydrolysis of p-nitrophenyl palmitate and fababean alkaline lipase. The significance of results from this study is discussed in relation to earlier work on lipid-degrading enzymes in fababeans.     

Nutritional and technological characteristics of new broad bean flaked products

The effects of the technological processes (soaking in water or alkaline solutions, drying, puree preparation) and the supplementation with maize flour on the nutritional value and on the organoleptic characteristics of broad bean (Vicia faba, L. major) flakes have been studied. Protein content was not affected by technological process. The addition of maize flour decreased the protein content of the final product depending on the amount of the maize flour added. Amino acid composition showed a decrease of tryptophan due to technological processing. Supplementation with maize flour improved the amino acid pattern and, except for tryptophan, the amount of essential amino acids in the flakes supplemented with 25% or more maize flour well compared with the provisional pattern by F.A.O. In vitro digestibility trials did not evidence significant changes due to technological processes or to integration of broad beans with maize flour. Broad bean toxic factors (vicine and convicine glycosides) were only slightly affected by the alkaline treatment of the flakes. Glycosides content decreased with the increasing supplementation with maize flour but the relationship was not linear. The organoleptic tests were positive for texture and taste, whereas the appearance of the products should be improved.     

Isolation of the egg weight depressing factor in fababeans (Vicia faba L. var. minor)

Two experiments were conducted to study the egg weight depressing effect of an ethanol-water extract of autoclaved fababean protein concentrate. Acetone fractionation of the extract produced four fractions, one of which (fraction H), significantly depressed egg weight. Further fractionation of fraction H produced two fractions; a highly water-soluble supernatant fraction and a fraction with low water solubility which was harvested as a white precipitate. Both the supernatant and the white precipitate, when added to diets in proportion to their relative yields, caused similar depressions in egg weight. The observation that there was a direct relationship between amounts of vicine-reactive material in the different fractions and the magnitude of egg weight depression indicates that the principal egg weight depressing factor in fababeans is vicine and/or convincine.     

Solubilization and Electrophoretic Characterization of the Great Northern Bean (Phaseolus vulgaris L.) Proteins

Protein content of dry beans (Phaseiolus vulguris L.) cultivar Great Northern, was 26.10% (dry weight basis). The isoelectirc pH of the NaCl extractable proteins was about 4.4. Several salts, NaOH, and HCl were employed to solubilize the Great Northern bean proteins. Amongst all the protein solubilizing agents, Na₂CO₃, K₂SO₄, sodium dodecyl sulfate (SDS), and NaOH at respective concentrations of 0.5, 5, 5% (all w/v), and 0.02N were found to be better protein solubilizers than the rest; solubilizing 93.6g of Lowry protein per 100g of Kjeldahl protein. Albumins and globulins accounted for 21.18 and 73.40% respectively, of the total bean proteins. Protein content of albumins, globulins, protein concentrates, and protein isolates was 81.68, 92.26, 85.44, and 92.43% (dry weight basis) respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the bean flour, albumins, globulins, protein concentrates, and protein isolates revealed the presence of 22, 14, 10, 14, and 11 subunits, respectively. The bean flour, albumin's, globulins, protein concentrates and protein isolates were characterized by the predominance of subunits with apparent molecular weights of 294,000, 266,000, 123,000, 146,000, and 135,000 daltons, respectively. Isoelectric focusing of the bean flour, albumins, globulins, protein concentrates, and protein isolates indicated 15, 13, 15, 16, and 11 subunits, respectively. Molecular sieve chromatography of the bean flour proteins, albumins, and globulins followed by SDS-PAGE was also employed to study the complexities of these proteins.     

Use of different dietary protein sources for lactating goats: milk production and composition as functions of protein degradability and amino acid composition

To establish the effect of the nature of four different protein sources [fababeans, 27.8% crude protein (CP); sunflower meal, 41.7% CP; corn gluten feed, 18.8% CP; and cottonseed, 18.3% CP] on milk protein production by goats, the ruminal degradation of these feeds was studied as was the amino acid (AA) composition of the original material and that of the undegradable fractions of the protein sources. Four diets were designed; 20% of their protein was supplied by each of the different sources. Four groups of 5 Granadina goats were used to study the utilization of these diets for milk production. No significant differences were observed in dry matter intake or milk production. The milk produced by goats fed the diet containing sunflower meal had the lowest protein concentration; the highest milk protein concentration was observed for goats fed the diet containing corn gluten feed. From a multivariate analysis, it was deduced that the quickly degradable protein fraction in the rumen and the ruminally undegradable protein fraction were the components of the protein sources most directly related to the milk protein produced. Given the similar AA profiles of the undegradable fractions of the different protein sources, the possible supplementation achieved from these ruminally undegradable fractions must be established by the amount of protein supplied regardless of AA composition.     

Effect of genotype and environment on the concentrations of starch and protein in, and the physicochemical properties of starch from, field pea and fababean

BACKGROUND: The effects of genotype and environment and their interaction on the concentrations of starch and protein in, and the amylose content and thermal and pasting properties of starch from, pea and fababean are not well known. RESULTS: Differences due to genotype were observed in the concentrations of starch and protein in pea and fababean, in the onset temperature (To) and peak temperature (Tp) of gelatinization of fababean starch, and in the pasting, trough, cooling and final viscosities of pea starch and fababean starch. Significant two‐way interactions (location × genotype) were observed for the concentration of starch in fababean and the amylose content, To, endothermic enthalpy of gelatinization (ΔH) and trough viscosity of fababean starch. Significant three‐way interactions (location × year × genotype) were observed for the concentration of starch in pea and the pasting, trough, cooling and final viscosities of pea starch. CONCLUSION: Differences observed in the concentrations of starch and protein in pea and fababean were sufficient to be of practical significance to end‐users, but the relatively small differences in amylose content and physicochemical properties of starch from pea and fababean were not. Copyright © 2011 Society of Chemical Industry