Degradation Of Sinigrin Research Articles

Enzyme systems involved in glucosinolate metabolism in Companilactobacillus farciminis KB1089

Cruciferous vegetables are rich sources of glucosinolates (GSLs). GSLs are degraded into isothiocyanates, which are potent anticarcinogens, by human gut bacteria. However, the mechanisms and enzymes involved in gut bacteria-mediated GSL metabolism are currently unclear. This study aimed to elucidate the enzymes involved in GSL metabolism in lactic acid bacteria, a type of gut bacteria. Companilactobacillus farciminis KB1089 was selected as a lactic acid bacteria strain model that metabolizes sinigrin, which is a GSL, into allylisothiocyanate. The sinigrin-metabolizing activity of this strain is induced under glucose-absent and sinigrin-present conditions. A quantitative comparative proteomic analysis was conducted and a total of 20 proteins that were specifically expressed in the induced cells were identified. Three candidate proteins, β-glucoside-specific IIB, IIC, IIA phosphotransferase system (PTS) components (CfPttS), 6-phospho-β-glucosidase (CfPbgS) and a hypothetical protein (CfNukS), were suspected to be involved in sinigrin-metabolism and were thus investigated further. We hypothesize a pathway for sinigrin degradation, wherein sinigrin is taken up and phosphorylated by CfPttS, and subsequently, the phosphorylated entity is degraded by CfPbgS. As expression of both pttS and pbgS genes clearly gave Escherichia coli host strain sinigrin converting activity, these genes were suggested to be responsible for sinigrin degradation. Furthermore, heterologous expression analysis using Lactococcus lactis suggested that CfPttS was important for sinigrin degradation and CfPbgS degraded phosphorylated sinigrin.

Antimicrobial and Cytotoxic Activities of Lepidium latifolium L. Hydrodistillate, Extract and Its Major Sulfur Volatile Allyl Isothiocyanate

The cultivated Lepidium latifolium L. was investigated to decipher its glucosinolate profile, antimicrobial, and cytotoxic activities. HPLC/ESI-MS analyses of the intact glucosinolates and GC/MS analysis of their hydrolysis products showed the presence of sinigrin (1), glucocochlearin (2), glucotropaeolin (3), and 4-methoxyglucobrassicin (4). Hydrodistillate, extract, and allyl isothiocyanate, the main volatile resulting from sinigrin degradation, showed antimicrobial activity against all eleven tested pathogenic and food spoilage bacteria and fungi, with highest effect observed against Candida albicans with MIC50 8 and 16 μg/mL. Hydrodistillate and extract showed the best cytotoxic activity on bladder cancer UM-UC-3 cell line during an incubation time of 24 h (IC50 192.9 and 133.8 μg/mL, respectively), while the best effect on glioblastoma LN229 cell line was observed after 48 h (IC50 110.8 and 30.9 μg/mL, respectively). Pure allyl isothiocyanate displayed a similar trend in cytotoxic effect on both cell lines (IC50 23.3 and 36.5 μg/mL after 24 h and 48 h, respectively).

Sinigrin degradation and G. pallida suppression in soil cultivated with brassicas under controlled environmental conditions

Inter-cropping mustard and oilseed radish in potato production has been shown to suppress potato cyst nematode (PCN) reproduction in field plots. This suppressive effect is mostly attributed to the release of glucosinolate (GSL) hydrolysis products such as isothiocyanates, following chopping and incorporation of the crop biomass. This study examined the degradation of sinigrin introduced into soil where Indian mustard and oilseed radish were cultivated and the effect on PCN suppression. Glucosinolate degradation was measured by introducing sinigrin into the soil samples pre-planting, pre- and post-incorporation of the brassicas, while the soil microbial activity was quantified as the concentration of fluorescein di-acetate (FDA) g−1 oven dry soil. Sinigrin was significantly (P<0.001) degraded pre- and post-incorporation of the brassicas cultivated in unsterilized soil. The total microbial activity increased significantly (P<0.001) pre- and post-incorporation of brassicas when compared with fallow controls. Unsterilized soils planted with oilseed radish resulted in a significant (P<0.027) suppression of G. pallida encysted eggs (30–35% mortality). Microbial activity correlated positively with G. pallida mortality (R2=0.94, P<0.001), but inversely with sinigrin degradation. This study has revealed that brassicas used for biofumigation may affect soil borne pests and pathogens not just after incorporation of the crop residues, but also during active growth of these plants.

Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7

This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304.

Identification of Proteins Possibly Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8.

This study was aimed to identify sinigrin-induced bacterial proteins potentially involved in the metabolism of glucosinolate in two glucosinolate-metabolising bacteria Lactobacillus agilis R16 and Escherichia coli VL8. Sinigrin (2 mM) was used to induce the proteins in both bacteria under anaerobic incubation for 8 h at 30 °C for L. agilis R16 and 37 °C for E. coli VL8 and the controls without sinigrin were performed. Allyl isothiocyanate and allyl nitrile as two degradation products of sinigrin were detected in sinigrin-induced cultures of L. agilis R16 (27% total products) and E. coli VL8 (38% total products) from a complete sinigrin degradation in 8 h for both bacteria. 2D gel electrophoresis was conducted to identify induced proteins with at least twofold increased abundance. Sinigrin-induced L. agilis R16 and the control produced 1561 and 1543 protein spots, respectively. For E. coli VL8, 1363 spots were detected in sinigrin-induced and 1354 spots in the control. A combination of distinct proteins and upregulated proteins of 32 and 35 spots in L. agilis R16 and E. coli VL8, respectively were detected upon sinigrin induction. Of these, 12 and 16 spots from each bacterium respectively were identified by LC-MS/MS. In both bacteria most of the identified proteins are involved in carbohydrate metabolism, oxidoreduction system and sugar transport while the minority belong to purine metabolism, hydrolysis, and proteolysis. This indicated that sinigrin induction led to the expressions of proteins with similar functions in both bacteria and these proteins may play a role in bacterial glucosinolate metabolism.

Influence of Temperature, Glucose, and Iron on Sinigrin Degradation by Salmonella and Listeria monocytogenes

Factors, including pH, temperature, glucose concentration, and iron compounds, affect the activity of plant myrosinase and, consequently, endogenous glucosinolate degradation. These factors also may affect glucosinolate degradation by bacterial myrosinase. Therefore, this study examined the effect of temperature (4 to 21°C), glucose (0.05 to 1.0%), and iron (10 mM ferrous or ferric) on sinigrin degradation by Salmonella or Listeria monocytogenes cocktails in Mueller-Hinton broth and the effect of sinigrin degradation on bacterial viability. The degradation of sinigrin by both pathogens increased with higher temperatures (21 > 10 > 4°C). Salmonella and L. monocytogenes cocktails hydrolyzed 59.1 and 53.2% of sinigrin, respectively, at 21°C up to 21 days. Both iron compounds significantly enhanced sinigrin degradation by the pathogens. On day 7, sinigrin was not detected when the Salmonella cocktail was cultured with ferrous iron or when the L. monocytogenes cocktail was cultured in Mueller-Hinton broth containing ferric iron. In contrast, ferric and ferrous iron inhibited the activity of 0.002 U/ml myrosinase from white mustard by 63 and 35%, respectively, on day 1. Salmonella and L. monocytogenes cocktails were able to degrade >80% of sinigrin at 0.05 and 0.1% glucose; however, 0.25 to 1.0% glucose significantly reduced sinigrin degradation. Although both pathogens significantly degraded sinigrin, the allyl isothiocyanate (AITC) recoverable was ≤6.2 ppm, which is not inhibitory to Salmonella or L. monocytogenes. It is probable that the gradual hydrolysis of sinigrin to form AITC either did not produce an inhibitory level of AITC or the AITC formed was unstable in the aqueous medium and rapidly decomposed to new compounds that were less bactericidal against the pathogens.

Influence of NaCl and NaNO3 on sinigrin hydrolysis by foodborne bacteria.

The glucosinolate sinigrin (SNG) is converted by endogenous plant myrosinase or by bacterial myrosinase-like activity to form the potent antimicrobial allyl isothiocyanate. In order to use SNG as a natural antimicrobial precursor in food, it became important to better understand the ability of bacteria to synthesize the enzyme(s) and understand factors influencing this synthesis at a constant SNG concentration. Eight spoilage, pathogenic, or starter culture bacteria were grown separately in medium containing individual or combined salts with SNG. SNG degradation by the bacteria and the formation of its major degradation product, allyl isothiocyanate, were followed for 12 days at 30 or 35°C. The bacterial strains varied in their ability to metabolize SNG, and this was enhanced by NaCl and/or NaNO(3). SNG hydrolysis took place after 4 days, and the greatest amount occurred by day 12. At 12 days, Escherichia coli O157:H7 showed the greatest capacity to hydrolyze SNG (45.3% degradation), followed by Staphylococcus carnosus (44.57%), while Pseudomonas fluorescens was not active against SNG. The ability of tested strains to metabolize SNG, in decreasing order, was as follows: Escherichia coli O157:H7 > Staphylococcus carnosus > Staphylococcus aureus > Pediococcus pentosaceus > Salmonella Typhimurium > Listeria monocytogenes > Enterococcus faecalis > Pseudomonas fluorescens.

Monitoring the enzymatic degradation of sinigrin from B. juncea meal using 1H NMR spectroscopy

A simple procedure for extracting and purifying sinigrin from Oriental mustard (Brassica juncea) meal using cold water was developed. Subsequently, the chemical degradation of sinigrin due to enzymatic activity was monitored using 1H NMR spectroscopy over the course of 30 h. The experimental findings show that sinigrin has been positively identified from B. juncea using 1H NMR spectroscopy and the prescribed preparation procedure. The results that have been presented also offer unambiguous evidence that 1H NMR spectroscopy can be used to detect changes in sinigrin concentration over time.

Glucosinolate Derivatives in Stored Fermented Cabbage

The research focused on the glucosinolate (GLS) breakdown products formed during the fermentation of cabbage. A relationship between the contents of degradation products in fermented cabbage and native GLS in raw cabbage was investigated. The effect of fermented cabbage storage on the contents of individual compounds was also assayed. Ascorbigen formed from one of the degradation products of glucobrassicin (indole GLS) was found to be a dominating compound in fermented cabbage. Irrespective of the time of fermented cabbage storage, the content of ascorbigen reached approximately 14 micromol/100 g. Neither the content of isothiocyanates, the major degradation products of aliphatic GLS, nor that of cyanides exceeded 2.5 microM. Storage of cabbage caused periodical increases and decreases in the contents of cyanides and consequent declines in the contents of isothiocyanates. The highest relative contents (expressed as a percentage of the native GLS content) of degradation products--ranging from >70 to 96%--were reported for the products of glucoraphanin degradation, whereas the lowest-- <5% --were reported for the products of sinigrin degradation.

Direct and simultaneous analysis of sinigrin and allyl isothiocyanate in mustard samples by high-performance liquid chromatography.

A reversed-phase HPLC method for the simultaneous determination of the glucosinolate sinigrin and its major degradation product allyl isothiocyanate (AITC) was developed and used for direct analysis of aqueous extracts from Oriental mustard (Brassica juncea L.) related materials (ground and cracked seeds, powders, and bran) and from soil samples. The lowest detection limit was 0.1 microg/mL for both sinigrin and AITC). The developed method was used to trace the degradation of sinigrin to AITC in aqueous extracts. One of the major advantages of this method is the complete estimation of sinigrin content. The simultaneous analysis of both sinigrin and AITC in a single run avoided the underestimation caused by separate analyses.

Factors affecting the dissolution and degradation of oriental mustard-derived sinigrin and allyl isothiocyanate in aqueous media.

Sinigrin, the predominant glucosinolate in the oriental mustard Brassica juncea, is mainly degraded upon the enzymatic action of myrosinase under normal conditions to give allyl isothiocyanate (AITC) in an aqueous media. Because AITC is considered to be the principal nematicidal ingredient in B. juncea, its stability in aqueous media is an important issue in achieving efficient nematode control. Pure sinigrin and AITC were found to be relatively stable in buffered water in the pH range of 5.00-7.00 but less stable at pH 9.00. Both sinigrin and AITC were more stable in soil water (supernatant of a 1:1 water/air-dried soil mixture) than in buffered water at the same pH range of 5.00-9.00. Sinigrin dissolved from the mustard bran or ground seed into water very quickly and was degraded by codissolved myrosinase to AITC. The AITC that formed from the degradation of sinigrin was found to be more stable in the soil water than in the buffered water. Buffer capacity was considered to be one of the factors that contributed to the stabilization of AITC in the soil water, but other unknown factors from both bran or seed and soil may also have contributed to the stabilization.

Degradation of sinigrin by Lactobacillus agilis strain R16

Forty-two lactobacilli were screened for their potential to degrade glucosinolate sinigrin. One of them, strain R16, demonstrated a high level of sinigrin degradation; it was identified as Lactobacillus agilis. The sinigrin degrading activity of L. agilis R16 could only be demonstrated when intact cells were used. The products of sinigrin degradation are allyl-isothiocyanate (AITC) and glucose (which is further fermented to dl-lactic acid), suggesting that myrosinase activity is involved. The activity was induced by the presence of sinigrin. Glucose inhibited the myrosinase activity, even in induced cells. Lactobacillus agilis R16 was able to grow on an extract of brown mustard seed and caused glucosinolate degradation.

1‐Cyano‐2,3‐Epithiopropane as the Primary Sinigrin Hydrolysis Product of Fresh Cabbage

ABSTRACTDichloromethane extracts of juices from fresh cabbages, including four known (Brutus, Galaxy, Bentley, Structon) and two unknown cultivars, were analyzed by X‐MS for the presence of sinigrin degradation products. Allyl isothiocyanate (AITC), which has been reported to be the important aroma compound of freshly disrupted cabbage was not detected in any of the dichloromethane extracts of cabbage juice. Instead, 1‐cyano‐2,3‐epithiopropane, which is one of the isomers of AITC, was the primary volatile compound in all cabbage extracts. AITC was detected in relative trace concentration only when cabbage juice was injected into the GC, using a wide bore, packed column. Thus, the relative importance of AITC and 1‐cyano‐2,3‐epithiopropane to the aroma of freshly disrupted cabbage should be considered.

Glucosinolate degradation by Aspergillus clavatus and Fusarium oxysporum in liquid and solid-state fermentation

Two fungal strains, Aspergillus clavatus II-9 and Fusarium oxysporum @ 149, proved to be capable of degrading sinigrin and sinalbin. During the degradation of sinigrin by whole cells of the Aspergillus strain, allylcyanide accumulated in the liquid incubation mixture. After a maximum concentration had been reached, the concentration of allylcyanide decreased as a result of its instability in the medium used. Incubation of cell-free extracts with sinigrin resulted in accumulation of glucose and allylisothiocyanate, suggesting that myrosinase is involved. Experiments with intact cells and cell-free extracts indicate the formation of an as yet unknown intermediate. When sinigrin was degraded by the Aspergillus strain in mustard seed meal under solid-state fermentation (SSF) conditions, no accumulation of allylcyanide or allylisothiocyanate was measured. Degradation of sinigrin by F. oxysporum @ 149 did not result in accumulation of intermediates, neither in liquid incubation mixtures nor in mustard seed meal under SSF conditions. Sinigrin was not degraded during incubation with cell-free extracts of F. oxysporum @ 149. Degradation of sinalbin by A. clavatus and F. oxysporum was measured during fermentation of yellow mustard seed meal under SSF conditions. Both fungi are useful for laboratory-scale SSF of mustard seed meal, thus opening new perspectives for a cost effective detoxification process for raw feed materials.

Effects of pH and Ferrous Ion on the Degradation of Glucosinolates by Myrosinase

During the enzymatic degradation of sinigrin, the release of glucose and the formation of allyl isothiocyanate via subsequent cleavage of the aglucon were studied in the presence or absence of ferrous ion over a pH range of 3.5 ~ 7.5. The liberation of glucose increased linearly with the time of enzymatic cleavage of sinigrin for the first 20 min at the pHs used, and no inhibition of glucose liberation was caused by ferrous ion at the concentration tried.On the other hand, the effects of ferrous ion on the formation of isothiocyanate changed greatly with changes of pH of the reaction medium. At pH 4.5 and 5.5, formation of isothiocyanate was strongly inhibited by ferrous ion, while such an effect of ferrous ion was notably depressed at pH 6.5 and disappeared at pH 7.5. These results were also confirmed by gas chromatographic analysis of the volatile cleavage products of the glucosinolates prepared from the seed meal of hakuran (an artificial variety of Brassica napus). From these results, it was suggested...

Effects of thiol compounds on the formation of nitriles from glucosinolates.

The effects of L-cysteine, reduced glutathione, dithiothreitol, cysteamine, thiobenzoate, thiomalate, and thiophenol on the enzymatic degradation of sinigrin were investigated in combination with ferrous ion at different pHs (5.0-7.5). Formation of allyl isothiocyanate was greatly inhibited by addition of both the thiol compounds and ferrous ion to the reaction medium at pH 5.0 or 6.5. Combined effects of the thiol compounds and ferrous ion on the formation of volatile products was also studied by gas chromatography using a mixture of allyl, 3-butenyl, and 4-pentenyl glucosinolates (1:4:1) as substrate. It was confirmed that the greater part of the volatile components produced in the presence of both the thiol compounds and ferrous ion was allyl, 3-butenyl, and 4-pentenyl cyanides and their corresponding cyanoepithioalkanes, even though the reaction medium was adjusted to pH 6.5. Similar results were obtained when silver sinigrate, an analogous model compound to the aglucon of sinigrin, was degraded by the addition of sodium chloride in the presence of both the thiol compounds and ferrous ion. The results suggest that in combination with ferrous ion, such common thiol compounds as L-cysteine and glutathione may be concerned in the production of nitriles from glucosinolates in natural systems, and that the acceleration of nitrile formation is probably attributed to much more rapid desulfuration than the progress of Lessen rearrangement of aglucon.