Identification of prenylated pterocarpans and other isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass spectrometry.

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the (1,3) A(+) retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H(2) O(x,y) RDA fragments, whereas its presence gave predominantly the common RDA fragments.

Interaction of flavan-3-ol derivatives and different caseins is determined by more than proline content and number of proline repeats.

Interactions of Type A and B flavan-3-ol dimers (procyanidins) and several monomeric flavan-3-ols, with α-casein and ß-casein, were investigated. Binding affinities measured were related to the ligands structure, including several properties (e.g. intrinsic flexibility (number of rotatable bonds) and hydrophobicity), and to the amino-acid composition of the caseins. A monomeric flavan-3-ol esterified with gallic acid (EGCG) had a five to ten times higher affinity to caseins compared to the non-galloylated dimeric flavan-3-ols. In this case, the larger number of rotatable bonds in EGCG might be accountable for this difference. Comparing flavan-3-ol dimers, intrinsic flexibility did not consistently promote interactions, as procyanidin A1 displayed a higher affinity to α-casein than the supposedly more flexible B-type dimers investigated. Despite its higher content of proline, compared to α-casein, ß-casein did not always have a higher affinity for the ligands investigated (e.g. no interaction with procyanidin A1 detected). These results suggest that more factors than proline content and the number of proline repeats govern phenolic-casein interactions.

Evaluation of the bitter-masking potential of food proteins for EGCG by a cell-based human bitter taste receptor assay and binding studies.

Epigallocatechin gallate (EGCG) has been ascribed to several health benefits, but its bitter taste influences the liking of products with high concentrations of this compound. ß-Casein, in particular, and several gelatins are known as strong binders of EGCG, contrary to ß-lactoglobulin. The current study aimed at relating the EGCG-binding characteristics of those proteins and their food-grade equivalents to their effects on reducing bitter receptor activation by EGCG in vitro and their bitter-masking potential in vivo. Also in the bitter receptor assay, ß-casein showed the strongest effect, with a maximum reduction of hTAS2R39 activation of about 93%. A similar potency was observed for Na-caseinate. ß-Lactoglobulin had little effect on bitter receptor activation, as expected based on its low binding affinity for EGCG. The bitter-masking potential of Na-caseinate was confirmed in vivo using a trained sensory panel. ß-Lactoglobulin also slightly reduced EGCG bitter perception, which could not be directly related to its binding capacity. The bitter receptor assay appeared to be a valid tool to evaluate in vitro the efficacy of food proteins as complexing agents for masking bitterness.

Efficacy of food proteins as carriers for flavonoids.

Enrichment of flavonoids in food is often limited by their off-tastes, which might be counteracted by the use of food proteins as carriers of flavonoids. Various milk proteins, egg proteins, and gelatin hydrolysates were compared for their binding characteristics to two flavan-3-ols. Among the proteins tested for their affinities toward epigallocatechin gallate (EGCG), ß-casein and gelatin hydrolysates, in particular fish gelatin, were found to be the most promising carriers with an affinity on the order of 10(4) M(-1). A flexible open structure of proteins, as present in random coil proteins, was found to be important. The saturation of binding observed at high flavonoid/protein ratios was used to estimate the maximal binding capacity of each protein. To reach a daily intake of EGCG that has been associated with positive health effects, only 519 mg of gelatin B and 787 mg of ß-casein were required to complex EGCG on the basis of their maximal binding capacity. When the absence of turbidity is taken into account, ß-casein prevails as carrier. Three selected proteins were further investigated for their binding potential of representative flavonoids differing in their C-ring structure. An increase in hydrophobicity of flavonoids was related to a higher affinity for proteins, and the presence of a gallic acid ester on the C-ring showed an overall higher affinity.