Determination of isothiocyanate-protein conjugates in milk and curd after adding garden cress (Lepidium sativum L.)

Abstract

Isothiocyanates (ITC) play an important role in health promotion and cancer prevention due to their anti-bacterial, anti-inflammatory, and anti-cancerogenic properties. However, ITC are highly reactive so that a reaction with further food components is very likely. For example, a reaction of ITC with nucleophilic amino acid side chains of proteins such as cysteine and lysine can occur, reducing the bioavailability of indispensable amino acids and protein functions may be altered. Therefore, it is of great interest to investigate the fate of ITC in the food matrix. Accordingly, the aim of the present study was to investigate the interaction of milk proteins and the ITC benzyl isothiocyanate (BITC) and allyl isothiocyanate (AITC) forming dithiocarbamates and thioureas in milk and curd. After incubating milk and curd with pure ITC or ITC-containing garden cress (Lepidium sativum L.), proteins were isolated, digested, and analyzed via LC-ESI-MS/MS as amino acid derivatives ("conjugates"). Protein conjugates of AITC and BITC were detected in all samples investigated. Further, the acidic pH value in curd favored the formation of dithiocarbamates over the formation of thioureas. Slightly acidic or neutral conditions like in fresh milk favored the formation of thioureas. The investigations also indicated that AITC shows a higher reactivity and dithiocarbamates are formed preferably, whereas incubation with BITC lead to less protein conjugates and the ratio of thioureas and dithiocarbamates was more balanced. In addition, amino acid modifications were often analyzed with indirect methods like measuring the decline of the amino acid residues. In this study, the modified amino acids were analyzed directly leading to more reliable results concerning the amount of modification.

Keywords: Allyl isothiocyanate; Benzyl isothiocyanate; Garden cress; Milk proteins; Protein conjugates.