The aim of this study was to investigate the impact of different metal salts in inactivation of myrosinase activity. no volatile products derived from glucosinolates. However, in the kohlrabi matrix, the strongest enzyme inhibition effect was observed for silver salt resulting in no volatile products, also both anhydrous Na2SO4 and saturated CaCl2 answer seem to be useful inhibitors in flavor studies. vegetables such as broccoli, brussels sprout, cabbage, cauliflower, kohlrabi, mustard, and radish; however it was also found in bacteria, fungi and even in the human gut microflora [8]. Glucosinolates in presence of myrosinase are prone to hydrolysis after tissue disruption (blending, trimming) into -d-glucose and an intermediate product (aglycon). This latter compound is usually spontaneously converted into isothiocyanates, nitriles, thiocyanates, epithionitriles, indoles, or 1,3-oxazolidine-2-thiones. The type of glucosinolate product obtained during hydrolysis purely depends on the reaction conditions and the original glucosinolate side-chain structure (aliphatic, aromatic, and indolic) [9,10]. Myrosinase is an enzyme that converts glucosinolates into glucosinolate degradation compounds, most of which are volatile and odor active. An active enzymatic reaction in the vegetable tissue is an analytical problem as it prospects to inaccurate results and large variations between samples. Screening of volatiles is not the only branch of analytical chemistry where blocking of thioglucosidase enzyme is usually important. Quantitative Rabbit polyclonal to ACTL8 analysis of metabolites (glucosinolates) in herb tissues requires the enzyme to be blocked, which prevents their further degradation during the analysis and prospects to deviations in the obtained results. The inactivation of the enzyme at an exact time is also important to increase the reproducibility of results obtained between laboratories. Currently, there are only few studies about the inactivation methods of myrosinase. The only well-known approach of deactivating this enzyme activity is usually pH changing [11], thermal treatment [12], pressure enzyme inactivation, or fusion of these latter two methods [13]. However, both methods have disadvantages; the first approach cannot be used simultaneously with the analysis of thermolabile compounds and pressure inactivation needs specific equipment that is unique and not available for all laboratories. The aim of this study was to investigate the impact of different metal salts in inactivation of myrosinase activity. The utilization of metal salts is simple to use, widely available, and relatively cheap. This knowledge will be useful for future research in flavor studies especially for identification and quantification of volatile compounds extracted from plant tissue and to improve analytical extraction protocols for glucosinolate-containing plants by increasing reproducibility when preparing samples for analysis. 2. Results 2.1. Preliminary Experiments with Commercial Thioglucosidase A preliminary experiment relied on analyzing volatile glucosinolate breakdown products in aqueous system. To select the most effective agent for the inactivation of thioglucosidase, the volatile products were determined by GC/MS system. The peak area represents the Gefitinib-based PROTAC 3 total ion current (TIC). As shown in Figure 1a not all of the investigated metal salts were effective at blocking myrosinase, resulting in a similar amount of obtained isothiocyanates as in the control test (H2O). Manganese ions proved to be completely ineffective at blocking the enzymatic reaction for both glucosinolates, glucotropeaolin, and synigrin. The adequate molar concentration of calcium and sodium ions and saturated solution of sodium chloride exhibited inhibition effect for synigrin, nevertheless they were ineffective in the case of blocking glucotropeaolin hydrolysis. Open in a separate window Figure 1 Volatile products of glucosinolates breakdown in miscellaneous enzyme inactivation media; sat.saturated solution; anh.anhydrous; a, b, c, d, ethe result of statistical analysis (one-way ANOVA Duncan test as post-hoc), values followed by the same letter in a.This knowledge will be useful for future research in flavor studies especially for identification and quantification of volatile compounds extracted from plant tissue and to improve analytical extraction protocols for glucosinolate-containing plants by increasing reproducibility when preparing samples for analysis. 2. flavor studies. vegetables such as broccoli, brussels sprout, cabbage, cauliflower, kohlrabi, mustard, and radish; however it was also found in bacteria, fungi and even in the human gut microflora [8]. Glucosinolates in Gefitinib-based PROTAC 3 presence of myrosinase are prone to hydrolysis after tissue disruption (blending, cutting) into -d-glucose and an intermediate product (aglycon). This latter compound is spontaneously converted into isothiocyanates, nitriles, thiocyanates, epithionitriles, indoles, or 1,3-oxazolidine-2-thiones. The type of glucosinolate product obtained during hydrolysis strictly depends on the reaction conditions and the original glucosinolate side-chain structure (aliphatic, aromatic, and indolic) [9,10]. Myrosinase is an enzyme that converts glucosinolates into glucosinolate degradation compounds, most of which are volatile and odor active. An active enzymatic reaction in the vegetable tissue is an analytical problem as it leads to inaccurate results and large variations between samples. Screening of volatiles is not the only branch of analytical chemistry where blocking of thioglucosidase enzyme is important. Quantitative analysis of metabolites (glucosinolates) in plant tissues requires the enzyme to be blocked, which prevents their further degradation during the Gefitinib-based PROTAC 3 analysis and leads to deviations in the obtained results. The inactivation of the enzyme at an exact time is also important to increase the reproducibility of results obtained between laboratories. Currently, there are only few studies about the inactivation methods of myrosinase. The only well-known approach of deactivating this enzyme activity is pH changing [11], thermal treatment [12], pressure enzyme inactivation, or fusion of these latter two methods [13]. However, both methods have disadvantages; the first approach cannot be used simultaneously with the analysis of thermolabile compounds and pressure inactivation needs specific equipment that is unique and not available for all laboratories. The aim of this study was to investigate the impact of different metal salts in inactivation of myrosinase activity. The utilization of metal salts is simple to use, widely available, and relatively cheap. This knowledge will be useful for future research in flavor studies especially for identification and quantification of volatile compounds extracted from plant tissue and to improve analytical extraction protocols for glucosinolate-containing plants by increasing reproducibility when preparing samples for analysis. 2. Results 2.1. Preliminary Experiments with Commercial Thioglucosidase A preliminary experiment relied on analyzing volatile glucosinolate breakdown products in aqueous system. To select the most effective agent for the inactivation of thioglucosidase, the volatile products were determined by GC/MS system. The peak area represents the total ion current (TIC). As shown in Figure 1a not all of Gefitinib-based PROTAC 3 the investigated metal salts were effective at blocking myrosinase, resulting in a similar amount Gefitinib-based PROTAC 3 of obtained isothiocyanates as in the control test (H2O). Manganese ions proved to be completely ineffective at blocking the enzymatic reaction for both glucosinolates, glucotropeaolin, and synigrin. The adequate molar concentration of calcium and sodium ions and saturated solution of sodium chloride exhibited inhibition effect for synigrin, nevertheless they were ineffective in the case of blocking glucotropeaolin hydrolysis. Open in a separate window Figure 1 Volatile products of glucosinolates breakdown in miscellaneous enzyme inactivation media; sat.saturated solution; anh.anhydrous; a, b, c, d, ethe result of statistical analysis (one-way ANOVA Duncan test as post-hoc), values followed by the same letter in a Duncan grouping are not significantly different, the subscript number and letter color are corresponding to the chart legend ( = 0.05); (n = 3); (a).