Optimizing isothiocyanate formation during enzymatic glucosinolate breakdown by adjusting pH value, temperature and dilution in Brassica vegetables and Arabidopsis thaliana

Abstract

Consumption of glucosinolate-rich Brassicales vegetables is associated with a decreased risk of cancer with enzymatic hydrolysis of glucosinolates playing a key role. However, formation of health-promoting isothiocyanates is inhibited by the epithiospecifier protein in favour of nitriles and epithionitriles. Domestic processing conditions, such as changes in pH value, temperature or dilution, might also affect isothiocyanate formation. Therefore, the influences of these three factors were evaluated in accessions of Brassica rapa, Brassica oleracea, and Arabidopsis thaliana. Mathematical modelling was performed to determine optimal isothiocyanate formation conditions and to obtain knowledge on the kinetics of the reactions. At 22 °C and endogenous plant pH, nearly all investigated plants formed nitriles and epithionitriles instead of health-promoting isothiocyanates. Response surface models, however, clearly demonstrated that upon change in pH to domestic acidic (pH 4) or basic pH values (pH 8), isothiocyanate formation considerably increases. While temperature also affects this process, the pH value has the greatest impact. Further, a kinetic model showed that isothiocyanate formation strongly increases due to dilution. Finally, the results show that isothiocyanate intake can be strongly increased by optimizing the conditions of preparation of Brassicales vegetables.

Figure 1. Enzymatic hydrolysis pathways of 2-propenyl glucosinolate (2Prop-GLS) to 2-propenyl isothiocyanate (2Prop-ITC), 3-butenenitrile (2Prop-CN) and 1-cyano-2,3-epithiopropane (CETP).

K₁–k₄ represent the ratios of the rate constants of the reactions. ESP: epithiospecifier protein.

Figure 2

Quadratic and reduced quadratic response surface models for 3-butenyl glucosinolate (3But-GLS) hydrolysis to (A) 3-butenyl isothiocyanate (3But-ITC; quadratic model), (B) 4-pentenenitrile (3But-CN; reduced quadratic model) and (C) 1-cyano-3,4-epithiobutane (CETB; reduced quadratic model) in Brassica rapa BR215. Displayed are relative values (%) formed from the respective breakdown product relative to the corresponding 3But-GLS. Red dots indicate design above predicted model and pink dots indicate those values that are below the predicted model.

Figure 3

Reduced quadratic and 2-factor interaction (2FI) response surface models for 2-propenyl glucosinolate (2Prop-GLS) hydrolysis to (A) 2-propenyl isothiocyanate (2Prop-ITC; reduced quadratic model), (B) 3-butenenitrile (2Prop-CN; reduced quadratic model) and (C) 1-cyano-2,3-epithiopropane (CETP; 2FI model) in Arabidopsis thaliana Hi-0. Displayed are relative values (%) formed from the respective breakdown product relative to the corresponding 2Prop-GLS. Red dots indicate design above predicted model and pink dots indicate those values that are below the predicted model.

Figure 4

Reduced quadratic response surface models for 4-(methylsulphinyl)butyl glucosinolate (4MSOB-GLS) hydrolysis to (A) 4-(methylsulphinyl)butyl isothiocyanate (4MSOB-ITC; reduced quadratic model) and (B) 5-(methylsulphinyl)pentanenitrile (4MSOB-CN; reduced quadratic model) in Brassica oleracea BroccoCress®. Displayed are relative values (%) formed from the respective breakdown product relative to the corresponding 4MSOB-GLS. Red dots indicate design above predicted model and pink dots indicate those values that are below the predicted model.

Figure 5

Kinetic models of the effect of dilution with (A) water at 22 °C, (B) artificial salivary fluid at 37 °C and (C) myrosinase-inactivated plant homogenate at 22 °C on 2-propenyl glucosinolate hydrolysis in white cabbage sprouts and of the effect of dilution with (D) water at 22 °C, (E) artificial salivary fluid at 37 °C and (F) myrosinase-inactivated plant homogenate at 22 °C on 4-(methylsulphinyl)butyl glucosinolate (4MSOB-GLS) hydrolysis in Brassica oleracea BroccoCress®. CETP: 1-cyano-2,3-epithiopropane; 4MSOB-CN: 5-(methylsulphinyl)pentanenitrile; 4MSOB-ITC: 4-(methylsulphinyl)butyl isothiocyanate; 2Prop-CN: 3-butenenitrile; 2Prop-ITC: 2-propenyl isothiocyanate. Black lines indicate the model for the epithionitrile, dashed lines represent the nitrile-model and dotted lines show the model for the respective isothiocyanate.