Decomposition of sinigrin by methanol/ammonia/water treatment in model systems and mustard (Brassica nigra L.) seed meal

 Sinigrin (allylglucosinolate) was stored at 20  °C, 40  °C, and 60  °C in solutions containing 10% ammonia in 95% methanol (CH₃OH/NH₃/H₂O). The individual samples were analyzed for their contents of sinigrin, its decomposition product allyl isothiocyanate (AITC) and other reaction products. The major reaction products were allyl thiourea, methyl ester of N-allyl thiocarbamoyl acid and allyl cyanide. A newly identified decomposition product of sinigrin was ethylcyanide. Defatted Brassica nigra seed meal was treated by the CH₃OH/NH₃/H₂O mixture to simulate the procedure used for rape and mustard seed meal detoxification. suspensions of the meal in the above mixture of solvents were heated at 20  °C, 40  °C and 60  °C, respectively. The major reaction products were the same as those arising from sinigrin. Minor products identified were 4-hydroxybenzyl isothiocyanate formed from sinalbin and 2-hydroxy-butenyl cyanide which arises from progoitrin. In all cases, AITC was very rapidly decomposed. Mechanisms explaining sinigrin decomposition are presented and discussed. Received: 3 May 1999     

Determination of sinigrin,sinalbin, allyl- and benzyl isothiocyanates by RP-HPLC in mustard powder extracts

A reversed phase-high performance liquid chromatography method was developed to quantify sinigrin, sinalbin, allyl isothiocyanate and benzyl isothiocyanate present in aqueous and freeze-dried yellow and Oriental (brown) mustard extract samples using two pre-treatment methods (autoclaving, boiling) to prevent degradation by myrosinase. The lowest detection limits for sinigrin and sinalbin were 0.05 mg/L and for allyl- and benzyl isothiocyanate were 2 mg/L. The methods developed make it possible to quantify both the glucosinolates (sinigrin, and sinalbin) and their hydrolysis products (allyl- and benzyl isothiocyanate) with the same mobile phase, and only require adjustment of the wavelength and a change in the ratio of the high performance liquid chromatography mobile phase solvents (tetrabutylammonium hydrogen sulphate and acetonitrile). The use of a single method yielded accurate and rapid results for the four compounds (sinigrin, sinalbin, allyl- and benzyl isothiocyanate). Autoclaving of both yellow and brown mustard powder before glucosinolate extraction did not consistently improve the amount of sinalbin and sinigrin recovered over boiling treatments because the thermal stability of myrosinase proved problematic in glucosinolate recovery. Nonetheless, the highest extract yields found were 4.06 g/100 g for sinigrin and 2.57 g/100 g for sinalbin, respectively, which represented over 94 g/100 g extract yield of sinigrin from the Oriental mustard powder.     

Purification of major glucosinolates from Brassicaceae seeds and preparation of isothiocyanate and amine metabolites

Consumption of Brassica vegetables containing glucosinolates is associated with decreased risk of cancer at several tissue sites. Studies of the relative potency and metabolism of the major dietary isothiocyanates have been hindered by limited availability of high‐purity isothiocyanates and their glucosinolate precursors. We describe a simple procedure for the preparation of glucosinolates sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin and gluconasturtiin from vegetable seeds. Glucosinolates were extracted from the seeds of mustard, oilseed, broccoli, cauliflower and winter cress. Purification by preparative anion exchange and reversed‐phase column chromatography gave the pure glucosinolates. Good separation was achieved without using ion‐pair reagents. Related isothiocyanates and amine degradation products were prepared by myrosinase‐catalysed hydrolysis of the corresponding glucosinolate and by organic synthesis, respectively. The compounds were characterized by analytical HPLC, ¹H NMR, ¹³C NMR, elemental analysis and mass spectrometry. Optimized conditions for detection and quantitation of these glucosinolates and metabolites by LC‐MS/MS are also given. Large‐scale glucosinolate preparation could be performed, giving amounts suitable for pharmacological evaluation in vivo. These methods will facilitate studies of the pharmacological activity and metabolism of dietary glucosinolates. Copyright © 2006 Society of Chemical Industry     

Inhibition of Listeria monocytogenes on bologna sausages by an antimicrobial film containing mustard extract or sinigrin

The ability of Listeria (L.) monocytogenes to convert glucosinolates into antimicrobial isothiocyanates was investigated. Mustard glucosinolates in pure (sinigrin) or extract forms (sinigrin, oriental; sinalbin, yellow mustard) were used in broth media and in a polyvinyl polyethylene glycol graft copolymer (PPG) packaging film with bologna to examine their value as antimicrobial precursors for the control of L. monocytogenes viability and extension of bologna shelf-life at 4 °C. During broth tests with deodorized (myrosinase-inactivated) mustard extracts (10 d at 20 °C) or with purified sinigrin (21 d at 20 °C) L. monocytogenes was only inhibited when exogenous myrosinase was added. None the less, the organism was able to hydrolyze almost half the pure sinigrin by 21 d in tests without added enzyme. Reductions in sinigrin levels were measured by reversed-phase liquid chromatography, and in the absence of L. monocytogenes or added myrosinase the glucosinolate was stable. When pure sinigrin, oriental or yellow mustard extracts were incorporated in PPG films containing 3, 5 and 6% (w/w) of the corresponding glucosinolate and used to package bologna inoculated with 4 log CFU/g L. monocytogenes, the pathogen became undetectable in bologna packed with the oriental mustard extract at 52 d storage and remained undetectable at 70 d. The yellow mustard extract was less inhibitory and the pure sinigrin was not antimicrobial. L. monocytogenes numbers reached >7 log CFU/g in the film and untreated controls at 17 d storage. At 35 d storage, samples packed with control film contained sufficient numbers of lactic acid bacteria (LAB) (>7 log CFU/g) to be considered spoiled, whereas treatments containing mustard or sinigrin remained <7 log CFU/g LAB for ≤ 70 d. L. monocytogenes played a key role in exerting control over its own viability in bologna by hydrolysis of the glucosinolate in the oriental mustard film, but other antimicrobials in treatments may have contributed.     

Analysis of volatile components during potherb mustard (Brassica juncea, Coss.) pickle fermentation using SPME-GC-MS

A method based on headspace solid-phase microextraction (HS-SPME) has been applied for the analysis of volatile components in fresh potherb mustard and their pickles with different pickling time. Hierarchical cluster analysis of the relative abundances of volatile compounds determined by GC-MS was used for the classification of the volatile components. As a result the glucosinolates enzymatically decomposed compounds account for the major proportion in volatile constituents. From the glucosinolates degradation products enzymatically via aglycone in fresh potherb mustard and their pickles, the result showed that the glucosinolates in potherb mustard contain allyl, butenyl, isobutanyl and phenylethyl groups in the side chain as substituents, which means that the glucosinolates are sinigrin, gluconapin, glucocochlearin and gluconasturtin, respectively. It can be also concluded from the experiments that the glucosinolates with allyl and phenylethyl groups as side chain in potherb mustard are predominant.     

Release of Allyl Isothiocyanate from Mustard Seed Meal Powder

Allyl isothiocyanate (AITC) is a wide‐spectrum antimicrobial compound found in mustard seeds, produced when their tissues are disrupted. The formation of AITC in mustard seed is mediated by the myrosinase enzyme which catalyzes the release of volatile AITC from a glucosinolate—sinigrin. Since water is a substrate in the reaction, humidity from the air can be used to activate the release of AITC from mustard seed. In this study, defatted and partially defatted mustard seed meals were ground into powders with particle size ranging from 5 to 300 μm. The mustard seed meal powder (MSMP) samples were enclosed within hermetically sealed glass jars wherein the headspace air was adjusted to 85% or 100% relative humidity at 5, 20, or 35 °C. Data from gas chromatography analysis showed that AITC release rate and amount increased with increasing relative humidity and temperature. Moreover, the release rate can be manipulated by particle size and lipid content of the MSMP samples. The amount of AITC released ranged from 2 to 17 mg/g MSMP within 24 h under the experimental conditions tested. In view of the antimicrobial properties of AITC, the mustard meal powder may be used as a natural antimicrobial material for extending the shelf life of food products.     

The thymol method for glucosinolate determination

This paper details experiments aimed at optimising the conditions for the determination of glucosinolates in rapeseed (Brassica napus L) using the colorimetric thymol procedure. Glucose could not be substituted for sinigrin when preparing a standard curve for glucosinolate determination unless a correction factor was employed. The value of this factor was found to depend on the concentration of sulphuric acid. Although the coloured complexes formed between thymol and either glucose or sinigrin were found to be unstable at 100°C, the sinigrin/ glucose colour ratio remained relatively constant for incubation times from 40 to 60 min. It was found necessary to increase the amount of thymol present to a final value of 0.23% to ensure complete reaction of glucosinolates. The revised method can be employed successfully for the analysis of glucosinolates in both seed and meal.     

Comparison of HPLC and GLC methodologies for determination of glucosinolates using reference material

 The reference material rapeseed of known glucosinolate composition, was analysed by HPLC and GLC and two commercially available glucosinolates (glucotropaeolin and sinigrin) were used as internal standards. The HPLC method enabled determination of 11 different glucosinolates (as desulphoglucosinolates) that occur in the rapeseed. Only seven glucosinolates (as trimethylsilylated desulphoglucosinolates) were separated by GLC. The latter method did not allow the determination of methylsulphinylalkyl glucosinolates (glucoiberin, glucoraphanin and glucoallysin) and did not separate optical isomers of 2-hydroxy-3-butenyl glucosinolate (progoitrin and epi-progoitrin). Statistical evaluation of data (t-test, F-test) revealed no significant differences between the tested methods at the 95% confidence level. The advantages and disadvantages of both widely used chromatographic methods are discussed. Received: 26 May 1997 / Revised version: 11 July 1997     

Comparison of HPLC and GLC methodologies for determination of glucosinolates using reference material

 The reference material rapeseed of known glucosinolate composition, was analysed by HPLC and GLC and two commercially available glucosinolates (glucotropaeolin and sinigrin) were used as internal standards. The HPLC method enabled determination of 11 different glucosinolates (as desulphoglucosinolates) that occur in the rapeseed. Only seven glucosinolates (as trimethylsilylated desulphoglucosinolates) were separated by GLC. The latter method did not allow the determination of methylsulphinylalkyl glucosinolates (glucoiberin, glucoraphanin and glucoallysin) and did not separate optical isomers of 2-hydroxy-3-butenyl glucosinolate (progoitrin and epi-progoitrin). Statistical evaluation of data (t-test, F-test) revealed no significant differences between the tested methods at the 95% confidence level. The advantages and disadvantages of both widely used chromatographic methods are discussed. Received: 26 May 1997 / Revised version: 11 July 1997     

Glucosinolates in brassica vegetables. Analysis of 22 varieties of brussels sprout (Brassica oleracea var. gemmifera)

The glucosinolate content of 22 cultivars of Brussels sprout have been determined. The levels of total glucosinolates, determined by methods based on glucose release and gas chromatography, were substantially higher than those reported by other workers for cabbage and Chinese cabbage. The major glucosinolates in Brussels sprout leaf material were identified as gluconapin, progoitrin, sinigrin and glucobrassicin, with the latter two generally predominating. Detailed analysis of the material from five different growing sites has shown considerable variation in the total content but surprisingly little difference in the relative proportions of the individual glucosinolates within each cultivar. The glucosinolates of the corresponding seed material differ significantly from that in leaf; in particular glucobrassicin is found in trace amounts only, and glucoerucin, glucotropaeolin and neoglucobrassicin are all present, in addition to sinigrin, progoitrin and gluconapin.     

Glucosinolates,volatile constituents and biological activities of Erysimum corinthium Boiss. (Brassicaceae)

Fresh leaves, roots and ripe seeds of Erysimum corinthium Boiss. (Brassicaceae) were investigated to uncover their glucosinolate contents through natural autolysis and exogenous myrosinase hydrolysis. The hydrolysis products were submitted to GC–MS analysis. Six glucosinolates were identified for the first time in this plant; namely, sinigrin, progoitrin, glucoiberin, 3-(methylcarbonyl)propyl glucosinolate, glucocheirolin and glucoerysolin. Glucocheirolin was the major compound accumulated in the seeds and progoitrin was the major compound in the roots, while 3-(methylcarbonyl)propyl glucosinolate was the major compound in the leaves. Other volatile constituents, e.g., terpenes and fatty acids esters were also identified. Seeds and leaves showed higher antimicrobial activity than roots. Seeds showed a marked cytotoxicity in vitro against colorectal, hepatic and Hela cell lines.     

Improved analysis of sinigrin in Ethiopian mustard

An ion‐pair reversed‐phase high‐performance liquid chromatography (HPLC) method was developed for the analysis of sinigrin in Ethiopian mustard (Brassica carinata A. Braun) seed and seed fractions. Separation was compared on several RP‐HPLC columns (Inertsil^® ODS‐4 C₁₈ and ZORBAX^® Eclipse XDB‐C₁₈) with an isocratic eluent containing 100% aqueous (aq.) tetramethylammonium bromide (10 mm , pH 5.0). Sinigrin retention was affected by HPLC variables including the type of ion‐pair reagent, buffer strength and pH, acetonitrile concentration, column temperature and eluent flow rate. Partial validation demonstrated this optimised chromatographic condition to be linear, accurate and precise. Multistage extraction using 70% (v/v) aq. methanol was more efficient than 50% (v/v) aq. acetonitrile. In addition, the matrix effect and recovery rate as well as processing efficiency of the analytical protocol were determined. This method is suitable for high throughput analysis of sinigrin in Ethiopian mustard seed and seed fractions.     

Glucosinolate profiles of international rapeseed lines (Brassica napus and Brassica campestris)

Seed samples from a range of international rapeseed lines (Brassica napus L and B campestris L) were analysed for glucosinolate content using high performance liquid chromatography (HPLC). This analytical method provided a total profile consisting of eight different glucosinolates, including those used to define the Canola standard. The data indicated that broad categories of distinct glucosinolate profiles existed among the seed lines analysed. Indole glucosinolate content, expressed as a percentage of total glucosinolate content, was higher in the low glucosinolate lines of B napus. The actual contents of all eight glucosinolates in the seed were variable, particularly gluconapin, glucobrassicanapin and progoitrin. The importance of this HPLC procedure in facilitating selection for high or low contents of individual glucosinolates is discussed.     

Supercritical Fluid Chromatography of Myrosinase Reaction Products in Ground Yellow Mustard Seed Oil

ABSTRACT: Escherichia coli O157:H7 can survive the dry fermented sausage manufacturing process. Compounds generated by enzymatic hydrolysis of glucosinolates present in ground mustard have shown potential antibacterial properties against E. coli O157:H7. In the present study, supercritical fluid chromatography (SFC) was used to investigate degradation products of 4-hydroxybenzyl glucosinolate (sinalbin), the major glucosinolate present in yellow mustard seed. Using SFC the concentration of the reactive component, 4-hydroxybenzyl isothiocyanate (PHBITC) was measured during the hydrolysis of sinalbin with myrosinase while imposing the temperature and pH regimen used for the fermentation phase of dry fermented sausage production. The PHBITC concentration grew to a maximum after 7 h and decreased rapidly. The concentration of 4-hydroxybenzyl alcohol increased rapidly to reach a maximum at 31 h and stayed at that level. The concentration of 4-hydroxybenzyl cyanate increased up to 24 h, then decreased to 65% of its maximum, and stayed constant. The stability of 4-hydroxybenzyl isothiocyanate in the presence of maltodextrin in this system was investigated. It was concluded that the major PHBITC loss in the mixture of the oil with maltodextrin occurred within the first 4 d of its storage. Practical Application: During the work 4-hydroxybenzyl isothiocyanate (PHBITC) was measured during the hydrolysis of sinalbin with myrosinase while imposing the temperature and pH regimen used for the fermentation phase of dry fermented sausage production. The results on PHBITC stability in maltodextrin presented provide precautionary information on the limited ability of this material to afford stability of PHBITC expected.     

The glucosinolates sinigrin and progoitrin are important determinants for taste preference and bitterness of Brussels sprouts

The glucosinolates, sinigrin and progoitrin, are related to the bitterness observed in Brussels sprouts. The role of these glucosinolates in taste preference for Brussels sprouts (Brassica oleracea var gemmifera) was studied with two different consumer taste panels and an analytical panel. Four different taste trials were conducted between 1989 and 1995 to study the role of sinigrin and progoitrin on taste preference and bitterness of sprouts. Sinigrin and progoitrin correlated negatively with taste preference of sprouts when their combined content was higher than 2·2 g kg⁻¹. Under these conditions the overall preference for different Brussels sprouts cultivars, in two different trial designs, was accounted for, respectively, 64 (Novartis Seeds BV) and 86% (TNO) by the contents of the glucosinolates sinigrin and progoitrin. In two additional trials, in which the combined content of sinigrin and progoitrin of all cultivars was below 2·2 g kg⁻¹, no relationship between taste preference and the content of both glucosinolates was observed. In the latter trials the content of sinigrin and progoitrin was found to be correlated with bitterness (r²_multiple = 0·67 and 0·93, respectively) as assessed by an analytical panel. The results of the taste trials show that both bitterness and taste preference for sprouts are correlated with the content of sinigrin and progoitrin. Taste preference for sprouts is only correlated with sinigrin and progoitrin when their combined content exceeds 2·2 g kg⁻¹, while bitterness is correlated with both glucosinolates for the whole studied content range. © 1998 Society of Chemical Industry.     

Urinary Mercapturic Acids as Markers for the Determination of Isothiocyanate Release from Glucosinolates in Rats Fed a Cauliflower Diet

Glucosinolates, found in plants of the genus Brassica, release physiologically active metabolites, such as isothiocyanates and nitriles, in the digestive tract under the action of plant or bacterial myrosinase. Isothiocyanates are excreted in the urine as their mercapturic acid derivatives. To study the influence of diet on isothiocyanate release, two groups of eight rats were offered either a cauliflower-based diet or a standard laboratory rat diet. After 10 days' adaptation to the diets release of allyl isothiocyanate (AITC) from a single oral dose of sinigrin, a glucosinolate commonly occurring in brassicas, was quantified. Each rat was dosed with 50 μmol of either sinigrin or AITC by stomach tube and urine was collected at 0, 2, 6, 24 and 48 h after administration. Three days later, rats which had previously received sinigrin were administered with AITC and vice versa. The concentrations of urinary mercapturic acids of isothiocyanates were determined by HPLC. Recoveries of the mercapturic acid derivative of AITC in each animal were used to estimate AITC production in sinigrin-dosed rats. Estimates of AITC release from sinigrin showed that a greater proportion of administered sinigrin was hydrolysed to AITC in cauliflower-fed rats than in rats fed a control diet (0·412 vs 0·134; P〈0·001). The approach should allow more rigorous investigation of the influence of dietary factors on glucosinolate hydrolysis in vivo. © 1997 SCI.     

Occurrence and Flavor Properties of Sinigrin Hydrolysis Products in Fresh Cabbage

Contrary to a published report, allyl isothiocyanate was confirmed as a major volatile sulfur compound along with 1-cyano-2,3-epithiopropane in dichloromethane extracts of freshly disrupted cabbage tissues resulting from thioglucosidase hydrolysis of allyl glucosinolate (sinigrin). Compared to authentic allyl isothiocyanate which had characteristic sharp, black mustard-like aroma notes, authentic 1-cyano-2,3-epithiopropane had weak, musty, sulfurous, and mustard-like aromas. Based on calculations of odor units (U_o) using data from the analysis of disrupted cabbage tissues, allyl isothiocyanate would contribute appreciably (to 245 U_o) while 1-cyano-2,3-epithiopropane would not likely contribute notably (<0.8 U_o) to fresh cabbage flavors.     

Content of p-Hydroxybenzylglucosinolate in seed meals of Sinapis alba as affected by heredity,environment and seed part

Seed meals of different cultivars and breeding lines of white mustard (Sinapis alba) grown at different localities and in different years were analysed for content of p-hydroxybenzylglucosinolate. The environmentally determined variation generally amounted to ± 12%. Cultivar differences were not significant in the material available but a much larger variation was found among individual plants. A statistical treatment of parent plant material and corresponding progenies gave a correlation coefficient of + 0·68 and a highly significant t² value. This indicates that the glucosinolate content of white mustard seed is mainly genetically determined. Analysis of samples from field experiments with fertilisers containing different amounts of sulphate revealed that the glucosinolate content was not influenced by a reduction in the amount of sulphate applied to heavy soils. The content of glucosinolate, while similar in cotyledons and hypocotyls, was, however, much lower in the seed coat.     

A simple spectrophotometric method for estimating total glucosinolates in mustard de-oiled cake

Glucosinolates are anti-nutritional factors present abundantly in the seed meal fraction of oilseed Brassica species. They are found in varying levels among different genotypes. Those genotypes containing less than 30 µmol/g are considered low/zero glucosinolate type and are preferred for edible purposes due to low pungency. Twenty two different genotypes were taken for the analysis of glucosinolates by spectrophotometry. A regression model was obtained using Ordinary Least Square technique which predicted a formula. Total glucosinolates (µmol/g) = 1.40 + 118.86 × A₄₂₅, where A₄₂₅ is the absorbance at 425 nm. The total glucosinolate content obtained by the prediction formula when compared with HPLC data showed a correlation coefficient of 0.942. This high correlation between the two data sets validated the developed methodology. This method also simplifies the estimation of total glucosinolates by excluding the use of HPLC or other sophisticated instruments.     

Consumer Acceptability and Sensory Profile of Cooked Broccoli with Mustard Seeds Added to Improve Chemoprotective Properties

Broccoli, a rich source of glucosinolates, is a commonly consumed vegetable of the Brassica family. Hydrolysis products of glucosinolates, isothiocyanates, have been associated with health benefits and contribute to the flavor of Brassica. However, boiling broccoli causes the myrosinase enzyme needed for hydrolysis to denature. In order to ensure hydrolysis, broccoli must either be mildly cooked or active sources of myrosinase, such as mustard seed powder, can be added postcooking. In this study, samples of broccoli were prepared in 6 different ways; standard boiling, standard boiling followed by the addition of mustard seeds, sous vide cooking at low temperature (70 °C) and sous vide cooking at higher temperature (100 °C) and sous vide cooking at higher temperature followed by the addition of mustard seeds at 2 different concentrations. The majority of consumers disliked the mildly cooked broccoli samples (70 °C, 12 min, sous vide) which had a hard and stringy texture. The highest mean consumer liking was for standard boiled samples (100 °C, 7 min). Addition of 1% mustard seed powder developed sensory attributes, such as pungency, burning sensation, mustard odor, and flavor. One cluster of consumers (32%) found mustard seeds to be a good complement to cooked broccoli; however, the majority disliked the mustard‐derived sensory attributes. Where the mustard seeds were partially processed, doubling the addition to 2% led to only the same level of mustard and pungent flavors as 1% unprocessed seeds, and mean consumer liking remained unaltered. This suggests that optimization of the addition level of partially processed mustard seeds may be a route to enhance bioactivity of cooked broccoli without compromising consumer acceptability.