Targeted inactivation of soybean proteinase inhibitors using zinc.

In this study the potential targeted use of zinc to inactivate proteinase inhibitors (PI) has been investigated as an alternative to the widely applied heat treatment used industrially for inactivation of PI. Zinc was utilized for the reduction of disulfide bonds leading to the structural changes in proteins, thus affecting the decreased affinity between PI and proteinases. The protein disulfide bond reduction mechanism was studied using a newly developed micellar electrokinetic capillary chromatography (MECC) with the glutathione redox reaction with dithiothreitol (DTT) as model system. This model proved efficient in monitoring the reduction of disulfide bonds in the Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). The use of zinc as a reductant resulted in a significant reduction of trypsin inhibitor activity (TIA) of 72% for KTI and 85% for BBI, highlighting zinc as a promising potential agent to reduce the activity of PI as an alternative to heat treatment.

Glutamine as an Ammonia Donor in Catabolism of the Glucosinolate, Sinalbin, in Biosynthesis of 4-Hydroxybenzylamine.

Amines synthesized by plants may be considered a dietary source of bioactive compounds, which are of interest due to possible health promoting effects. Developing Sinapis alba sprouts are known to produce 4-hydroxybenzylamine, but the reaction mechanism has not yet been established. We propose here a suggested metabolic pathway for the formation of 4-hydroxybenzylamine in S. alba plants. The catabolic sequence starts with a reaction between l-glutamine (Gln) as ammonia donor and 4-hydroxybenzyl carbocation, the enzymatic catalyzed hydrolysis product from sinalbin (4-hydroxybenzylglucosinolate). The suggested reactions are compared with alternative plant metabolic reactions used in the biosynthesis of biogenic amines.

Non-enzymatic transformations of dietary 2-hydroxyalkenyl and aromatic glucosinolates in the stomach of monogastrics.

Monogastric animals exhibit different biological responses to structurally diverse glucosinolates and their transformation products, depending on the dietary levels. The transformations of 2-hydroxyalkenyl and aromatic glucosinolates were examined in vitro under gastric conditions, ex vivo in ligated porcine stomachs and in vivo in a rat model. Intact glucosinolates were almost completely transformed in vitro within 1 h at pH 3 (73-88%) and at pH 5 (97-100%) upon addition of Fe2+ ranging from two-fold molar excess. Glucosinolate transformations reached 78-99% when incubated ex vivo in ligated porcine stomachs. Rat in vivo feeding trials showed major reductions (81-84%) in the intact glucosinolate contents upon passage through the gastrointestinal (GI) tract. Non-enzymatic transformations of glucosinolates occur in the stomach, where pH and the level of Fe2+ are primary determinants. This is the first study to show a complex formation between iron-progoitrin and iron-sinalbin, facilitating the transformation into nitriles and thionamides.

Piglet performance and physiological effects linked to reduced glucosinolate transformations in feed products based on rapeseed pressed cakes.

The nutritional quality of rapeseed press cakes (RPCs) in piglet feed is closely linked to its digestibility and the content of glucosinolates. This study investigates the significance of intact glucosinolate (glc) levels and degree of glc transformations on piglets performance. Four different RPCs were made from a low glc (11 µmol/g seed DM) containing B. napus L. seed variety Lioness (RPC-LW, RPC-LXW, RPC-LC, RPC-LCD). RPC made from the variety Excalibur containing the upper level of glc (24 µmol/g seed DM) of double rapeseed and produced at higher and prolonged temperature (RPC-UXW) served as negative control, while soya bean protein concentrate served as positive control. Piglets (8 kg) were fed ad libitum diets balanced for RPC protein content, with RPC inclusion of 84-98 g/kg (day 0-14) and 151-178 g/kg (day 15-50). Glc transformation was reduced from 42% to 24% (7.3-4.2 µmol/g RPC) when the temperature input was lowered in the warm pressing of oil, while the glc loss was less pronounced (17%) when cold pressing was applied. The following feed pelleting process further reduced Glc concentration from 11% to 40% in warm-pressed RPCs and 54 to 85% in cold-pressed RPCs. The RPC products replaced soya bean protein without any negative effects on performance, except for piglets served cold-pressed RPC, which had a reduction in average daily weight gain (ADG) (5%-7%, p < 0.05, Day 15-50). RPC in the feed led to increased liver weight in all piglets (p = 0.026). This may point at long-term effects from feeding with RPC. Intestinal absorption of intact glcs was proven by their detection in urine. In conclusion, warm-pressed RPC can be used as feed for piglet, while the presence of active myrosinase may have a negative effect on performance and cakes should either be included in lower amounts than used in the present study (18%) or include myrosinase inactivation before use.

Potential of cultivar and crop management to affect phytochemical content in winter-grown sprouting broccoli (Brassica oleracea L. var. italica).

BACKGROUND: Variety and crop management strategies affect the content of bioactive compounds (phenolics, flavonoids and glucosinolates) in green broccoli (calabrese) types, which are cultivated during summer and autumn in temperate European climates. Sprouting broccoli types are morphologically distinct and are grown over the winter season and harvested until early spring. Thus they show considerable potential for development as an import substitution crop for growers and consumers during the 'hungry gap' of early spring. The present study investigated the effect of variety and management practices on phytochemical content in a range of sprouting broccoli varieties. RESULTS: Yields were significantly higher in white sprouting broccoli varieties. Levels of phenolics and flavonoids were in the range 81.64-297.65 and 16.95-104.80 mg 100 g⁻¹ fresh weight, respectively, depending on year and cultivar, and were highest in variety 'TZ 5052' in both years. In-row spacing did not affect flavonoid content. Phenolic and flavonoid content generally increased with increasing floret maturity and levels were high in edible portions of the crop. Crop wastes (leaf and flower) contained 145.9-239.3 and 21.5-116.6 mg 100 g⁻¹ fresh weight total phenolics and flavonoids, respectively, depending on cultivar, tissue and year. Climatic factors had a significant effect on phenolic and flavonoid content. Levels of total and some individual glucosinolates were higher in sprouting broccoli than in the green broccoli variety 'Ironman'. CONCLUSION: Levels of total phenolics, flavonoids and glucosinolates are higher in sprouting than green broccoli types. Sprouting broccoli represents an excellent source of dietary bioactive compounds.

Screening for anti-nutritional compounds in complementary foods and food aid products for infants and young children.

A range of compounds with negative nutritional impact - 'anti-nutrients' - are found in most plant foods. The contents of anti-nutrients in processed foods depend on the ingredients and processing. Anti-nutrients in complementary foods for children can have a negative impact on nutritional status. The aim of this study was to screen complementary foods from developing countries for the anti-nutritional compounds, phytate, polyphenols, inhibitors of trypsin and chymotrypsin, and lectins. Commercial products based on whole grain cereals were included as a 'worst-case' scenario for anti-nutrient exposure in Europe. Contents of minerals (iron, zinc and calcium), in which absorption or utilisation is affected by anti-nutrients, were analysed. Thirty-six products representing foods used in food aid programmes, local blended foods, fortified instant porridges and 'baby foods' were analysed. The content of minerals indicated that the fortification of a number of products did not meet the declared levels of iron, zinc and calcium. The phytate content ranged from 68 to 1536 mg/100 g, confirming a persistent problem of high levels of phytate in processed cereal- and legume-based products. The phytate : Fe molar ratio exceeded the recommended level of <1.0 in 32 of the 36 products. The total polyphenols varied from 1.3 to 9.3 mg gentisic acid equivalents g(-1) . Screening low-molecular weight soluble polyphenols may be more relevant in complementary foods than total polyphenolic compounds. Trypsin and chymotrypsin inhibitors and lectins were found in residual amounts in most products, indicating efficient degradation by heat processing. However, young infants and malnourished children may have reduced pancreatic function, and upper limits for residual trypsin inhibitors are needed.

The use of environmental metabolomics to determine glyphosate level of exposure in rapeseed (Brassica napus L.) seedlings.

Metabolic profiling in plants can be used to differentiate between treatments and to search for biomarkers for exposure. A methodology for processing Ultra-High-Performance Liquid Chromatography-Diode-Array-Detection data is devised. This methodology includes a scheme for selecting informative wavelengths, baseline removal, retention time alignment, selection of relevant retention times, and principal component analysis (PCA). Plant crude extracts from rapeseed seedling exposed to sublethal concentrations of glyphosate are used as a study case. Through this approach, plants exposed to concentrations down to 5 µM could be distinguished from the controls. The compounds responsible for this differentiation were partially identified and were different from those specific for high exposure samples, which suggests that two different responses to glyphosate are elicited in rapeseed depending on the level of exposure. The PCA loadings indicate that a combination of other metabolites could be more sensitive than the response of shikimate to detect glyphosate exposure.

Ingestion of broccoli sprouts does not improve endothelial function in humans with hypertension.

UNLABELLED: Ingestion of glucosinolates has previously been reported to improve endothelial function in spontaneously hypertensive rats, possibly because of an increase in NO availability in the endothelium due to an attenuation of oxidative stress; in our study we tried to see if this also would be the case in humans suffering from essential hypertension. METHODS: 40 hypertensive individuals without diabetes and with normal levels of cholesterol were examined. The participants were randomized either to ingest 10 g dried broccoli sprouts, a natural donor of glucosinolates with high in vitro antioxidative potential, for a 4 week period or to continue their ordinary diet and act as controls. Blood pressure, endothelial function measured by flow mediated dilation (FMD) and blood samples were obtained from the participants every other week and the content of glucosinolates was measured before and after the study. Measurements were blinded to treatment allocation. RESULTS: In the interventional group overall FMD increased from 4% to 5.8% in the interventional group whereas in the control group FMD was stable (4% at baseline and 3.9% at the end of the study). The change in FMD in the interventional group was mainly due to a marked change in FMD in two participants while the other participants did not have marked changes in FMD. The observed differences were not statistically significant. Likewise significant changes in blood pressure or blood samples were not detected between or within groups. Diastolic blood pressure stayed essentially unchanged in both groups, while the systolic blood pressure showed a small non significant decrease (9 mm Hg) in the interventional group from a value of 153 mm Hg at start. CONCLUSION: Daily ingestion of 10 g dried broccoli sprouts does not improve endothelial function in the presence of hypertension in humans. TRIAL REGISTRATION: Clinicaltrials.gov NCT00252018.

Fe2+-catalyzed formation of nitriles and thionamides from intact glucosinolates.

The ratio of isothiocyanates to nitriles formed upon the hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate-containing plants and materials. A micellar electrokinetic capillary chromatography (MECC) method was used to study the nonenzymatic Fe2+-catalyzed transformation of glucosinolates. At room temperature, pH 5, and in the presence of only 2 molar excess of Fe2+ all glucosinolate was degraded in 24 h. At all molar excess Fe2+ tested, nitriles were the major compounds formed. Thionamides were also formed from glucosinolates that contained a side chain hydroxylated at C-2; in this case, trace amounts of oxazolidine-2-thione were also detected. The presence of Fe3+ had no effect. The nonenzymatic Fe2+-catalyzed transformation of glucosinolates involves the binding of Fe2+ to the glucosinolate to form a complex.

A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds.

Myrosinase is a beta-thioglucosidase glucohydrolase that catalyses the hydrolysis of the thioglucoside bond in glucosinolates, allelochemicals present in Brassicaceous plants. These isoenzymes have been found to form complexes with other proteins; however, traditional isolation procedures involving ammonium sulphate precipitation and/or ion exchange chromatography do not allow for the isolation of these complexes. The present paper reports a fast and gentle procedure for the isolation of myrosinases in the complex form. Partial purification by Con A affinity chromatography followed by Sephadex G-200 gel filtration allowed for the isolation of myrosinase complexes from seeds of Brassica carinata, B. oleracea var. capitata, B. napus and Sinapis alba. Myrosinases in the Brassicas formed complexes of different molecular weight (500-600 kDa, 270-350 kDa and 140-200 kDa) whereas in seeds of S. alba it was only possible to isolate and detect 140-200 kDa complexes. In all species the complexes were formed by isoenzymes with isoelectric points between 4.8 and 5.6 and in some cases up to 6.8. SDS-PAGE confirmed that the myrosinase isoenzymes were composed by several protein subunits of molecular weights ranging between 10 and 110 kDa. The relative amount and enzymatic activity of the myrosinase complexes varied amongst the species studied. The isolation of myrosinase complexes in their native form is of great importance for the study of the hydrolysis of glucosinolates under autolysis conditions.

Determination of shikimate in crude plant extracts by micellar electrokinetic capillary chromatography.

A method based on micellar electrokinetic capillary chromatography (MECC) has been developed for the determination of shikimate in water and crude plant extracts. The analytes are separated in a cholate-taurine buffer by MECC at pH 7.3 and measured by direct UV detection at 206 nm. Shikimate showed linearity up to 12.5 mM, with a squared correlation coefficient (r(2)) of 0.9997. The method has concentration limit of detection (cLOD) and concentration limit of quantification (cLOQ) at 24.4 and 67.8 microM, respectively, corresponding to detection in the femtomol range. The number of theoretical plates (N) was estimated to 245,000 for the optimized system using a capillary with an effective length of 560 mm. The method was tested on plant samples by measuring the shikimate content in leaves of rapeseed plants grown in hydroponic solutions containing the herbicide glyphosate, a well-known inhibitor of the shikimate pathway. In crude extracts of these plants, shikimate was found to accumulate in the leaves, confirming earlier reports of shikimate as a potential biomarker for glyphosate treatment. The method now developed was also able to detect shikimate-3-phosphate, but this compound was not accumulated in glyphosate inhibited plants as found for shikimate.

Micellar electrokinetic capillary chromatography--synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates.

A micellar electrokinetic capillary chromatography (MECC) method has been developed for monitoring the myrosinase catalysed hydrolysis of 2-hydroxy substituted glucosinolates and the simultaneous formation of the corresponding degradation products (oxazolidine-2-thiones (OZTs) and nitriles). Glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate) was chosen as the model glucosinolate owing to the difficulties in determining hydrolysis rates of this type of substrates in traditional UV-assays. The method was afterwards validated with glucobarbarin ((2S)-2-hydroxy-2-phenylethylglucosinolate) and progoitrin ((2R)-2-hydroxybut-3-enylglucosinolate). Aromatic glucosinolates without a 2-hydroxy group in their side chains, such as glucotropaeolin (benzylglucosinolate) and gluconasturtiin (phenethylglucosinolate) were also tested. Formation of the glucosinolate hydrolysis products was monitored simultaneously at 206 nm and 230 nm. This allowed estimation of the extinction coefficient of the OZT derived from glucosibarin, which was found to be 18,000 M(-1) cm(-1) and 12,000 M(-1) cm(-1) at 206 nm and 230 nm, respectively. The developed method has limit of detection of 0.04 mM and 0.06 mM and limit of quantification of 0.2 mM and 0.3 mM for the glucosibarin derived OZT and nitrile, respectively. Linearity of the glucosinolate concentration was examined at six concentration levels from 2.5 mM to 100 mM and at 206 nm a straight line (R(2)=0.9996) was obtained. The number of theoretical plates (N) at the optimal system conditions was 245,000 for the intact glucosibarin, 264,000 for the OZT and 252,000 for the nitrile.

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

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

Analysis of reducing carbohydrates by reductive tryptamine derivatization prior to micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography method for determination of low molecular weight carbohydrates (dp 1-2) with an unbound carbonyl group as in aldoses or other reducing carbohydrates has been developed. Reductive amination of aldoses on the carbonyl group using tryptamine introduced a chromophor system to the carbohydrates enabling their sensitive UV detection at 220 nm and identification based on the indole group using diode array detection. Twelve carbohydrates including pentoses (d-ribose, l-arabinose, and d-xylose), hexoses (d-glucose, d-mannose, and d-galactose), deoxy sugars (l-rhamnose and l-fucose), uronic acids (d-glucuronic acid and d-galacturonic acid), and disaccharides (cellobiose and melibiose) are included in the study, using d-thyminose (2-deoxy-d-ribose) as the internal standard. Detection of all 12 carbohydrates is performed within 30 min. Linearity with correlation coefficients from 0.9864 to 0.9992 was found in the concentration range of 25-2500 micromol/L for all carbohydrates; the relative standard deviation on the migration times was between 0.27 and 0.80 min, and limits of quantification and limits of determination were in the picomole range.

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).