ß-Lactoglobulin as nanotransporter for allicin: Sensory properties and applicability in food.

The thiosulfinate allicin is a labile, bioactive compound of garlic. In order to enrich allicin in a functional food, a delivery system which stabilises the compound and masks its intense flavour is necessary. In the present study allicin was covalently bound to the whey protein ß-lactoglobulin and the incorporation of this transporter in a food matrix was tested. The sensory properties of the pure functional ingredient as well as of an enriched beverage were characterised by quantitative descriptive analysis. The concentration of volatile compounds was analysed by headspace gas chromatography-mass spectrometry. The garlic-related organoleptic properties of garlic powder were significantly improved by the binding of allicin in combination with spray drying. After purification of the modified ß-lactoglobulin the garlic taste and smell were barely perceptible. ß-Lactoglobulin modified with allicin provided a stable functional ingredient that can be used to enrich a broad range of food products.

Influence of fermentation on glucosinolates and glucobrassicin degradation products in sauerkraut.

A systematic investigation was carried out on the influence of fermentation on glucosinolates and their degradation products from fresh raw cabbage, throughout fermentation at 20 °C and storage at 4 °C. Glucosinolates were degraded dramatically between Day 2 and 5 of fermentation and by Day 7 there was no detectable amount of glucosinolates left. Fermentation led to formation of potential bioactive compounds ascorbigen (13.0 µmol/100 g FW) and indole-3-carbinol (4.52 µmol/100g FW) with their higher concentrations from Day 5 to Day 9. However, during storage indole-3-carbinol slowly degraded to 0.68 µmol/100 g FW, while ascorbigen was relatively stable from Week 4 until Week 8 at 6.78 µmol/100 g FW. In contrast, the content of indole-3-acetonitrile decreased rapidly during fermentation from 3.6 to 0.14 µmol/100 g FW. The results imply a maximum of health beneficial compounds after fermentation (7-9 days) in contrast to raw cabbage or stored sauerkraut.

Influence of postharvest UV-B treatment and fermentation on secondary plant compounds in white cabbage leaves.

The influence of postharvest UV-B on its own and in combination with fermentation (e.g. sauerkraut production) on formation and degradation of bioactive compounds was investigated in white cabbage, processed according to traditional Chinese fermentation methods. The pattern of polyphenols was affected by postharvest UV-B: Newly formed coumaroylglycoside, feruloylglycoside, caffeoylglycoside (up to 1 mg/g dry matter; 4 days) and quercetintriglycoside (0.4-0.5 mg/gdm; 4 days) might be related to postharvest increase in enzyme activity in the biosynthesis. Decreasing contents were observed for the glucosinolates glucobrassicin and 4-methoxyglucobrassicin, but the formation of the degradation products dihydroascorbigen and dihydro-4-methoxyascorbigen, which might be related to cell shrinking as mechanical damage. Fermentation resulted in deglycosidation of hydroxycinnamic acids. Newly generated cinnamic acid from coumaric acid aglycone was detected in fermented plant material combined with UV-B (50 µg/g). Glucosinolates and dihydroascorbigens were completely degraded. This study shows exemplary UV-B as a supplemental step to improve the nutritional quality of processed plants.

ß-Lactoglobulin as nanotransporter--Part I: Binding of organosulfur compounds.

The binding reaction of allicin and diallyl disulfide with ß-lactoglobulin and the influence of pH value and protein denaturation on this reaction have been examined in the present study. Regardless of the structural similarity of both the organosulfur compounds, their binding behavior was significantly different. Both ligands were covalently bound by the free thiol group of the protein, whereas the affinity for allicin was significantly higher. In addition, diallyl disulfide was non-covalently bound. The binding reaction of both ligands was very sensitive to the pH value during incubation. The optimal pH range was between pH 8.0 and 9.0. Protein denaturation increased the reaction rate and reduced the number of binding sites for allicin, whereas the number of non-covalent binding sites increased for diallyl disulfide. Based on these findings, it can be proposed that the covalent modification of ß-lactoglobulin functions as a specific transporter stabilizing allicin or diallyl disulfide.

ß-Lactoglobulin as nanotransporter--Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide.

The whey protein ß-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified ß-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified ß-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to ß-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.

Characterization of the covalent binding of allyl isothiocyanate to ß-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry.

Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with ß-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace-water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.

Synthesis and Nrf2-inducing activity of the isothiocyanates iberverin, iberin and cheirolin.

Numerous studies have reported a potent induction of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent gene expression by the isothiocyanate sulforaphane. However, little is known regarding the Nrf2-inducing activities of the lower structure-related sulforaphane homologues, such as iberverin, iberin and cheirolin, which exhibit different sulfur oxidation states. Therefore, in this study we synthesized the isothiocyanates iberverin, iberin and cheirolin with a high yield and purity and determined their Nrf2-inducing activity in NIH3T3 fibroblasts. Iberverin, iberin and cheirolin significantly induced Nrf2 nuclear translocation. The increase in nuclear Nrf2 levels was accompanied by a significant increase in heme oxygenase 1 (HO-1) and γ-glutamylcysteine synthetase (γGCS) mRNA and protein levels. Overall, iberverin, iberin and cheirolin exhibited a similar potency to sulforaphane in inducing Nrf2-dependent gene-expression. Furthermore, our data suggest that the induction of Nrf2 by iberverin, iberin and cheirolin may have occurred via an extracellular signal-related kinase (ERK)-dependent signal-transduction pathway.