Perennial Baki™ Bean Safety for Human Consumption: Evidence from an Analysis of Heavy Metals, Folate, Canavanine, Mycotoxins, Microorganisms and Pesticides.

Global food production relies on annual grain crops. The reliability and productivity of these crops are threatened by adaptations to climate change and unsustainable rates of soil loss associated with their cultivation. Perennial grain crops, which do not require planting every year, have been proposed as a transformative solution to these challenges. Perennial grain crops typically rely on wild species as direct domesticates or as sources of perenniality in hybridization with annual grains. Onobrychis spp. (sainfoins) are a genus of perennial legumes domesticated as ancient forages. Baki™ bean is the tradename for pulses derived from sainfoins, with ongoing domestication underway to extend demonstrated benefits to sustainable agriculture. This study contributes to a growing body of evidence characterizing the nutritional quality of Baki™ bean. Through two studies, we investigated the safety of Baki™ bean for human consumption. We quantified heavy metals, folate, and canavanine for samples from commercial seed producers, and we quantified levels of mycotoxins, microorganisms, and pesticides in samples from a single year and seed producer, representing different varieties and production locations. The investigated analytes were not detectable or occurred at levels that do not pose a significant safety risk. Overall, this study supports the safety of Baki™ bean for human consumption as a novel pulse crop.

Antibacterial and Antibiofilm Effects of Allelopathic Compounds Identified in Medicago sativa L. Seedling Exudate against Escherichia coli.

In this study, the allelopathic properties of Medicago sativa L. (alfalfa) seedling exudates on the germination of seeds of various species were investigated. The compounds responsible for the allelopathic effects of alfalfa were identified and characterized by employing liquid chromatography ion mobility high-resolution mass spectrometry. Crude exudates inhibited the germination of seeds of all various plant species tested. Overall, nine compounds in alfalfa were identified and quantified. The most predominant compounds were a hyperoside representing a flavonoid glucoside, the non-proteinogenic amino acid canavanine, and two dipeptides, identified as H-Glu-Tyr-OH and H-Phe-Glu-OH. The latter corresponds to the first finding that dipeptides are exuded from alfalfa seedlings. In addition, the antibacterial and antibiofilm activities of alfalfa exudate and its identified compounds were elucidated. Both hyperoside and canavanine revealed the best antibacterial activity with minimum inhibitory concentration (MIC) values that ranged from 8 to 32 and 32 to 256 µg/mL, respectively. Regarding the antibiofilm action, hyperoside and canavanine caused a decline in the percentage of E. coli isolates that possessed a strong and moderate biofilm-forming potential from 68.42% to 21.05% and 31.58%, respectively. Studies on their inhibiting effects exhibit that these major substances are predominantly responsible for the allelopathic and antimicrobial effects of the crude exudates.

d-canavanine affects peptidoglycan structure, morphogenesis and fitness in Rhizobiales.

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here, we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyses production of dl-CAN from l-CAN, which is produced by many legumes. Importantly, d-CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that d-CAN alters dramatically the PG structure of Rhizobiales (e.g., Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens, we demonstrated that the detrimental effect of d-CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.

ß-N-Methylamino-L-Alanine Toxicity in PC12: Excitotoxicity vs. Misincorporation.

The implication of ß-N-methylamino-L-alanine (BMAA) in the development of neurodegenerative diseases worldwide has led to several investigations of the mechanism, or mechanisms, of toxicity of this cyanobacterially produced amino acid. The primary mechanism of toxicity that was identified is excitotoxicity, with a second possible mechanism, the misincorporation of BMAA into the primary protein structure and consequent cell damage, having been more recently reported. However, studies on excitotoxicity and misincorporation have been conducted independently and there are therefore no data available on the relative contribution of each of these mechanisms to the total toxicity of BMAA. The rat pheochromocytoma cell line PC12 is an ideal model for a study of this type, as glutamate receptor expression is modified by cell differentiation, which can be affected by exposure to nerve growth factor. In this study, the PC12 cell line was evaluated as a model to study BMAA toxicity via the two proposed mechanisms: excitotoxicity and protein misincorporation. BMAA and canavanine treatment of cultures of PC12 were evaluated for depolarization of the mitochondrial membrane. In canavanine-treated cultures, this was evident after 9 days of treatment and was attributed to the primary mechanism of canavanine toxicity, protein misincorporation. However, no membrane depolarization was observed for BMAA-treated cultures even after 21 days of continuous treatment at 500 µM. Short-term exposure to both BMAA and canavanine resulted in a slight increase in necrosis in undifferentiated cells that was prevented in canavanine-treated cultures by co-incubation with arginine, but not in BMAA-treated cultures by co-incubation with serine. A slight increase in apoptosis was observed in undifferentiated cells treated with either BMAA or glutamate, and ROS production increased in glutamate-treated cells. However, the excitotoxicity was less pronounced than reported in previous studies with neuronal cells. In contrast, apoptosis was greatly increased in both BMAA- and glutamate-treated cells after differentiation and resulting mGluR1 increase, indicating that excitotoxicity is the main, if not only, mechanism of toxicity in PC12.

Presence of L-canavanine in Hedysarum alpinum seeds and its potential role in the death of Chris McCandless.

OBJECTIVE: For the past 2 decades there has been vigorous disagreement over the purported toxicity of Hedysarum alpinum seeds, and whether the consumption of such seeds was a factor in the 1992 death of Chris McCandless, the subject of the book Into the Wild. Our objective was to confirm or disprove the presence of L-canavanine (a nonprotein amino acid known to induce systemic lupuslike symptoms in humans) in H alpinum seeds. METHODS: Liquid chromatography-tandem mass spectrometry analysis of H alpinum seeds was performed. RESULTS: Our analysis confirmed the presence of L-canavanine in H alpinum seeds and demonstrated that it is a significant component of the seeds, with a concentration of 1.2% (weight/weight), roughly half of that found in Canavalia ensiformis. CONCLUSIONS: The data led us to conclude it is highly likely that the consumption of H alpinum seeds contributed to the death of Chris McCandless.

Determination of L-canavanine and other free amino acids in Vicia disperma (Fabaceae) seeds by precolumn derivatization using diethyl ethoxymethylenemalonate and reversed-phase high-performance liquid chromatography.

A method for determination of the non-protein amino acid l-α-amino-γ-(guanidinooxy)-n-butyric acid (L-canavanine) and other free amino acids in Vicia disperma is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification were 0.15 and 0.50 µM, respectively. The method has a high intra- (RSD=0.35%) and inter-repeatability (RSD=2.86%), and a remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Quantification of canavanine, 2-aminoethanol, and cyanamide in Aphis craccivora and its host plants, Robinia pseudoacacia and Vicia angustifolia: effects of these compounds on larval survivorship of Harmonia axyridis.

The cowpea aphid Aphis craccivora that infests the black locust Robinia pseudoacacia shows toxicity to its predator, the multicolored Asian ladybird beetle, Harmonia axyridis. In contrast, the same aphid species that infests the common vetch, Vicia angustifolia, is suitable prey for H. axyridis larvae. Previously, it was reported that the toxicity of A. craccivora infesting R. pseudoacacia was due to canavanine and 2-aminoethanol, but there was some doubt about the toxicity of these compounds and their concentrations in the aphids. In the present study, we determined the concentrations of cyanamide, canavanine, and 2-aminoethanol in A. craccivora infesting the two host plants. In the extracts of A. craccivora that infested either of the host plants, canavanine was undetectable, and 2-aminoethanol was detected at the concentration of 3.0-4.0 µg/g fresh weight. Cyanamide was detected in the extract of A. craccivora that infested R. pseudoacacia (7.7 µg/g fresh weight) but not in that infesting V. angustifolia. The toxicity of canavanine, 2-aminoethanol, and cyanamide was evaluated against H. axyridis larvae in a bioassay by using an artificial diet containing these compounds at various concentrations. Cyanamide exhibited 10-100 times stronger toxicity than canavanine and 2-aminoethanol. These results indicate that the toxicity is at least partly due to cyanamide, which is present in the toxic A. craccivora that infests R. pseudoacacia but absent from the non-toxic A. craccivora that infests V. angustifolia.

[Identification and functional characterization of genes induced by seed exudates in Azorhizobium caulinodans ORS571].

OBJECTIVE: To identify genes induced by plant seed exudates in Azorhizobium caulinodans ORS571. METHODS: Using promoterless kanamycin resistance gene (Km(r)) on transposon as reporter gene and seed exudates as inducers, we screened genes of interest from transposon insertion mutants libraries. We streaked mutants on TY solid medium with Km, and another with Km and seed exudates correspondingly. If Km(r) is inserted into a gene that can be induced by plant signals, Km(r) will possibly express at the same time. Thus, mutants were selected that can grow on medium with Km and exudates, rather than on medium with Km. RESULTS: We identified a lysE family gene named asiE in strain Azc0 that can be induced by seed exudates and further analysis indicated that the inducing substance is canavanine (CAN). lacZ transcriptional fusion of asiE confirmed that its expression increased by ten-fold or so under the induction of CAN. Besides, lysE gene in four different species of Rhizobia can be induced by CAN. lysE mutants are all sensitive to CAN treatment whereas wild type are resistant. CONCLUSION: The existence of LysE can make rhizobia better survived in the rhizosphere and may play an important role in early stage of interaction between rhizobia and host plant.

Canavanine content in sword beans (Canavalia gladiata): analysis and effect of processing.

The amino acid canavanine is a potentially toxic constituent of leguminous seeds. The aim of the present study was to determine the ability of different processing methods to reduce canavanine in sword beans (Canavalia gladiata). For this purpose a method for the detection and quantification of canavanine was developed using reversed-phase high-performance liquid chromatography of the dabsylated derivatives. The recovery of canavanine using this method was 88-91%. Optimum extraction of canavanine from raw and processed beans was obtained by addition of hot water prior to overnight soaking. The results obtained with this method agree well with previously published values for raw seeds. The method is sensitive, specific and can successfully be applied to the detection of canavanine in legumes. Overnight soaking and boiling in excess water followed by decanting gave the most pronounced reduction in canavanine content (around 50%), followed by boiling and decanting excess water (34%). Roasting as used in this study and autoclaving were less effective in reducing the canavanine content.

Simplified qualitative method for canavanine in seeds and sprouts.

The major stored nitrogen compound in alfalfa seeds is canavanine. To identify this nonprotein amino acid from seed extract and sprout water, a qualitative micro-thin-layer chromatography method was developed. Successful separation and identification was achieved using microsilica plates, a 70:30 ethyl alcohol-water solvent system, and 1% ammonium disodium pentacyanoammineferrate II for color development. This quick method was used to identify canavanine (sensitivity 50 microg) from irradiated and nonirradiated alfalfa and clover seed extracts and alfalfa sprout water. Broccoli and radish seed extracts were negative for canavanine. This simple method is useful to track the release and decrease of canavanine in the sprout water.

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry.

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.

Características químicas y nutricionales del grano de cinco (5) genotipos de Canavalia ensiformis / Chemical and nutritional characteristics from grains of five genotypes of Canavalia ensiformis

Se realizó la caracterización del grano de cinco genotipos de Canavalia ensiformis, mediante la determinación de la composición química proximal, presencia de factores antinutricionales (Canavalia y título hemaglutinante) y digestibilidad in vitro. Los genotipos estudiados fueron: Original, Yaracuy, Valle de la Pascua, U-02 y Tovar. Los resultados de la composición química proximal mostraron diferencias significativas entre los genotipos a excepción de la humedad, encontrándose los siguientes promedios: Proteína: 31,37 por ciento, fibra: 8,10 por ciento: cenizas: 2,93 por ciento, grasa: 2,97 por ciento y humedad: 11,68 por ciento. El contenido de canavanina de los genotipos fue variable, oscilando los valores entre 2,02 a 4,86 por ciento presentando el genotipo u-02 el valor mayor, respocto al título hemaglutinante varió entre +2 y +5. La digestibilidad proteica in vitro de las harinas arrojó diferencias signifacativas entre los genotipos, la cual varió entre 47,51 por ciento y 51,84 por ciento, valores muy por debajo al mostrado por la caseína (97,3 por ciento)

[Chemical and nutritional characteristics from grains of five genotypes of Canavalia ensiformis]. / Características químicas y nutricionales del grano de cinco (5) genotipos de Canavalia ensiformis.

This study evaluated the raw meals from grains of five genotypes of Canavalia ensiformis, by means of the chemical composition, the presence of antinutritional factors (Canavanine and hemaglutination activity) and in vitro protein digestibility. The genotypes studied were: Original, Yaracuy, Tovar, Valle de la Pascua and U-02. The results of chemical composition, showed significance difference between them, except moisture content, found the following average values: Protein 31.37%, fiber: 8.10%, ash: 2.93% and moisture: 11.68%. The canavanine content of the genotypes was variable oscillating between 2.02 and 4.86%, the genotype U-02 presented the higher value, respect to the hemaglutination title changed between: +2 and +5. The in vitro protein digestibility of the raw meals showed significance differences between the genotype, it changed between 47.51% and 51.84%, these values were lower than the casein (97.3%).

[Flour from seeds of Canavalia ensiformis L (DC), raw, stored in an alkaline medium, autoclaved or extracted, in diets for growing pigs]. / Harina de granos de Canavalia ensiformis L (DC) cruda, almacenada en medio alcalino, autoclavada o extruída, en dietas para cerdos en crecimiento.

There were made four nutrition experiments using pork in growing process, with an approximate weight of 14.2 kilograms. In each experiment, it was made a substitution in equals parts of corn flour and soy flour for raw Canavalia flour or processed through alkaline storage, autoclaved or extrusion. In the first three experiments, the substitution level of raw Canavalia (RC), stored in alkaline environment (CAMA) or autoclave (CA) were: 0, 5, 10 and 15%. In the fourth one, the including levels of Canavalia Extruida were: 0, 7.5 and 15%. The raw Canavalia as the Extruida drastically reduced the pork's growth. The Canavalia flour autoclaved (121 degrees C/15 psi/90 min) substantially improved the animal answers, even though the growth in all the substitution levels were lower than the one observed in the original portion. The storage of the beans in alkaline environment, made possible better productive behavior in the animals, and it didn't observe differences (p < 0.01) to increase the witness weight, at the substitution level or 5%. The pork's answer to the Canavalia toxin was manifested in the first term, by a drastic reduction in the voluntary consumption of foods. As a whole, the results indicated that none of the methods used were effective to eliminate or to minimize the toxic effects in the raw Canavalia over the productive behavior of growing pork.

Canavanine analysis of alfalfa extracts by high performance liquid chromatography using pre-column derivatization.

The purpose of this project was to isolate and quantitate canavanine in three different alfalfa samples because it has been hypothesized that alfalfa and therefore canavanine ingestion may exacerbate systemic lupus erythematosus (SLE) in humans. Ten alfalfa tablets were ground into a fine powder, and 100 mg was extracted for canavanine. The extracted canavanine was derivatized prior to quantitation by high performance liquid chromatography. The canavanine content of alfalfa tablets averaged 25.3 and 33.8 micrograms/g depending on lot number and extraction method. Because ten alfalfa tablets are recommended daily, this would provide approximately 0.18 mg of canavanine. Whether daily ingestion of canavanine exacerbated SLE in two patients consuming 15 and 8 alfalfa tablets (0.27 and 0.15 mg, respectively) is uncertain and warrants further investigation.

Elimination of toxicity from diets containing alfalfa seeds.

Cynomolgus macaques were fed autoclaved alfalfa seeds for up to 1 yr. There were no humoral signs of a syndrome resembling systemic lupus erythematosus. The data are in contrast to those previously reported in monkeys fed raw alfalfa seeds, in which a systemic lupus erythematosus-like syndrome was induced in a shorter interval. The autoclaved seeds retained antihypercholesterolaemic effects.

A specific arginase-based assay for L-canavanine in leguminous plants.

A convenient method for the analysis of free L-canavanine in leguminous plants is described. Canavanine was specifically hydrolyzed to canaline and urea by the enzyme arginase (EC 3.5.3.1). The resulting amino-oxy functions of canaline were measured based on their ability to bleach the yellow colour of pyridoxal 5'-phosphate. Canavanine in the seeds of Canavalia ensiformis was determined with this method.

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.