RESUMO
An obstacle in the application of many "health ingredients" is their alleged off-flavor. We used a combination of chemical, sensory, and biological analyses to identify the bitter components in citrus peel-derived polymethoxyflavone preparations, claimed to be functional in the lowering of cholesterol. Nobiletin (56-81%) and tangeretin (10-33%) were found to be the main bitter components. Using in vitro receptor assays, hTAS2R14 was shown to be the main bitter receptor involved in their perception, with EC50 values of 14 and 63 µM, respectively. Our analysis provided several routes for off-flavor reduction. Purification is an option because a purified, single PMF species proved to be considerably less bitter upon application in emulsified foods, due to limited solubility in the aqueous phase. A second route, also demonstrated in vivo, is C5-specific demethoxylation, in line with the finding that 5-desmethylnobiletin does not activate hTAS2R14. A third route could be the use of TAS2R14 antagonists. As a proof of principle, several antagonists, with IC50 values ranging from 10 to 50 µM, were identified.
Assuntos
Citrus/química , Flavonas/química , Aromatizantes/química , Polifenóis/química , Receptores Acoplados a Proteínas G/química , Paladar/fisiologia , Cromatografia Líquida de Alta Pressão , Estrutura MolecularRESUMO
The human bitter taste receptor hTAS2R39 can be activated by many dietary (iso)flavonoids. Furthermore, hTAS2R39 activity can be blocked by 6-methoxyflavanones, 4'-fluoro-6-methoxyflavanone in particular. A structure-based pharmacophore model of the hTAS2R39 binding pocket was built using Snooker software, which has been used successfully before for drug design of GPCRs of the rhodopsin subfamily. For the validation of the model, two sets of compounds, both of which contained actives and inactives, were used: (i) an (iso)flavonoid-dedicated set, and (ii) a more generic, structurally diverse set. Agonists were characterized by their linear binding geometry and the fact that they bound deeply in the hTAS2R39 pocket, mapping the hydrogen donor feature based on T5.45 and N3.36, analogues of which have been proposed to play a key role in activation of GPCRs. Blockers lack hydrogen-bond donors enabling contact to the receptor. Furthermore, they had a crooked geometry, which could sterically hinder movement of the TM domains upon receptor activation. Our results reveal characteristics of hTAS2R39 agonist and bitter blocker binding, which might facilitate the development of blockers suitable to counter the bitterness of dietary hTAS2R39 agonists in food applications.
Assuntos
Receptores de Superfície Celular/metabolismo , Software , Sítios de Ligação , Desenho de Fármacos , Flavanonas/química , Flavanonas/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/antagonistas & inibidoresRESUMO
The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.
Assuntos
Aspergillus/enzimologia , Biotecnologia/métodos , Fungos Mitospóricos/enzimologia , Peroxidase/genética , Aspergillus/genética , Aspergillus/crescimento & desenvolvimento , Northern Blotting , Southern Blotting , Fermentação , Heme/metabolismo , Fungos Mitospóricos/genética , Peroxidase/metabolismo , Plasmídeos , Regiões Promotoras GenéticasRESUMO
Many (dietary) bitter compounds, e.g. flavonoids, activate bitter receptor hTAS2R39 in cell-based assays. Several flavonoids, amongst which some flavanones, are known not to activate this receptor. As certain flavanones are known to mask bitter taste sensorially, flavanones might act as bitter receptor antagonists. Fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds occurring in green tea. Three flavanones showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4'-fluoro-6-methoxyflavanone, 6,3'-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the full antagonist 4'-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate.
Assuntos
Catequina/análogos & derivados , Flavanonas/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Bioensaio , Cálcio/metabolismo , Sinalização do Cálcio , Catequina/antagonistas & inibidores , Catequina/química , Catequina/farmacologia , Flavanonas/química , Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-Atividade , Paladar/fisiologia , Chá/química , TransgenesRESUMO
Many flavonoids and isoflavonoids have an undesirable bitter taste, which hampers their use as food bioactives. The aim of this study was to investigate the effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 and to predict their structural requirements to activate these receptors. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, among which 58 ligands were overlapping. Their activation threshold values varied over a range of 3 log units between 0.12 and 500 µM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure-activity relationships. The 2D models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 (or hTAS2R39) were determined by 3D-pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. The additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behaviors on the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in the prediction of their potential bitterness. By understanding the "bitter motif", introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.
Assuntos
Flavonoides/metabolismo , Isoflavonas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Paladar , Motivos de Aminoácidos , Sítios de Ligação , Flavonoides/química , Humanos , Isoflavonas/química , Cinética , Modelos Moleculares , Ligação ProteicaRESUMO
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.
Assuntos
Caseínas/metabolismo , Catequina/análogos & derivados , Receptores de Superfície Celular/metabolismo , Paladar , Caseínas/química , Catequina/química , Catequina/metabolismo , Feminino , Humanos , Cinética , Masculino , Ligação Proteica , Receptores de Superfície Celular/químicaRESUMO
The aim of this study was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 µM on hTAS2R14 and hTAS2R39 and EC(50) values of 29 and 49 µM, respectively. In addition, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar toward most isoflavonoid aglycones. By trend, threshold values were slightly lower on hTAS2R14. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely, glycitin, genistin, acetylgenistin, and malonylgenistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than fewer hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the determined bitter receptor threshold values, whereas those of other soy isoflavones are around or below their respective threshold value. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.
Assuntos
Flavonoides/metabolismo , Genisteína/metabolismo , Glycine max/química , Extratos Vegetais/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética , Ativação Transcricional , Linhagem Celular , Flavonoides/química , Genisteína/química , Humanos , Cinética , Extratos Vegetais/química , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
We report the expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori. Fragments encoding V(HH)s were cloned in a suitable Aspergillus expression vector and transformants secreting V(HH) fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V(HH)s were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V(HH)s were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.