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Sensory characteristics of human milk : Association between mothers' diet and milk for bitter taste
Mastorakou, Dimitra ; Ruark, Angelica ; Weenen, Hugo ; Stahl, Bernd ; Stieger, Markus - \ 2019
Journal of Dairy Science 102 (2019)2. - ISSN 0022-0302 - p. 1116 - 1130.
bitterness - breastfeeding - human milk - maternal diet - sensory perception

It is unknown how consumption of bitter foods and beverages in the maternal diet influences sensory properties of fresh human milk. The aims of this study were (1) to determine the sensory characteristics of fresh human fore and hind milk, (2) to establish relationships between sensory properties and composition of fresh human milk, and (3) to assess the relationship between bitterness of the maternal diet and human milk. Twenty-two lactating mothers generated sensory terms to describe perception of their milk and received training on sensory attribute intensity rating. Mothers kept a 24-h food diary followed by a sensory self-assessment of their fore and hind milks. The odor of human fresh milk was described as neutral, creamy, and sweet, taste as sweet and bitter, and mouthfeel as thin, watery, smooth, and fatty. Sweetness was equivalent to 1.53 g of sucrose/100 mL and was not significantly different between fore and hind milk. Fore milk was significantly less creamy, less fatty, thinner, more watery, and lower in vanilla flavor intensity than hind milk. Carbohydrate content of human milk was positively correlated with sweetness and glutamic acid content with umami. The bitterness of the diet consumed 24 h before lactation was moderately positively correlated with bitterness of fore milk, but not hind milk. We conclude that the consumption of bitter foods may influence the bitterness of human fore milk, which may be another way for breastfed children to learn to accept bitter vegetables and, hence, develop healthier food preferences.

Intrinsic bitterness of flavonoids and isoflavonoids and masking of their taste activity
Roland, W.S.U. - \ 2014
Wageningen University. Promotor(en): Harry Gruppen; Gerrit Smit, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789461738530 - 188
flavonoïden - isoflavonoïden - bitterheid - receptoren - chemische structuur - flavonoids - isoflavonoids - bitterness - receptors - chemical structure

Many flavonoids and isoflavonoids have been associated with beneficial health effects. Therefore, consumption of (iso)flavonoid-rich food products, and enrichment of foods with (iso)flavonoids is becoming increasingly popular. However, several (iso)flavonoids have been reported as bitter. Consequently, their incorporation in (or fortification of) foods can introduce (or enhance) bitterness. Hence, debittering strategies are demanded.

Some (iso)flavonoids have unknown taste properties, as they have never been incorporated in high levels in food products. For other (iso)flavonoids, contradictory findings on bitterness have been made in sensory tests. Therefore, objective tests are necessary to identify which (iso)flavonoids contribute to bitterness of a food product. An objective tool to study bitterness is a cell-based bitter taste receptor assay. Twenty-five different bitter taste receptors (hTAS2Rs) occur on the human tongue, each of which has been introduced in a separate human embryonic kidney (HEK)293 cell line. With these, the “intrinsic bitterness” of a compound can be investigatedin vitro. Intrinsic bitterness is the capacity of a compound to activate bitter taste receptors, uncoupled from cross-modal interactions and interactions with salivary proteins and oral mucosa. The aim of this research was to study the intrinsic bitterness of a large set of (iso)flavonoids and to investigate structural requirements for (iso)flavonoids to activate the bitter receptors identified. A subsequent aim was the investigation of different debittering strategies by the use of the bitter receptor assay.

Chapter 1provides an overview of flavonoids and isoflavonoids with respect to their structural classification, sensorial properties and occurrence as dietary compounds. Taste perception and the mode of action of bitter taste receptors are introduced. The measurement of bitter receptor activation in vitro is explained, as well as strategies to reduce bitter receptor activation, and bitter taste in general. A state-of-the-art overview of all 25 bitter taste receptors is given with respect to known agonists and antagonists.

The aim of Chapter 2was 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 EC50 values of 29 and 49 µM, respectively. Besides, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar towards most isoflavonoid aglycones. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely glycitin, genistin, acetyl genistin, and malonyl genistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than less hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the bitter receptor threshold values determined, whereas those of other soy isoflavones are around or below their respective threshold values. 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.

In Chapter 3, the intrinstic bitterness of (iso)flavonoids, which can hamper their use as food bioactives, was investigated further.The effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 was tested, and their structural requirements to activate these receptors were predicted. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, amongst which 58 ligands were overlapping. Their activation threshold values varied over a range of three 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 and hTAS2R39 were determined by 3D-pharmacophore models to be composed of two (for hTAS2R14) or three (for hTAS2R39) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. An 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 behavior towards 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 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.

Bitter receptor hTAS2R39 is activated by many different classes of bitter compounds, amongst which (iso)flavonoids. Nevertheless, several flavanones are known to mask bitter taste sensorially, andnot all flavanones reported in Chapter 3 activated hTAS2R39. For that reason, in Chapter 4, fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds present in green tea.Three compounds 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 most potent 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, suggesting a non-competitive orthosteric mechanism. The bitter receptor blockers identified might not be applicable to food products. Nevertheless, they create insight into structural requirements, which might lead to other, more suitable, blockers.

Chapter 5investigates another strategy to reduce bitterness, namely complexation of bitter flavonoids with food proteins. The binding characteristics of the bitter tea compound epigallocatechin gallate (EGCG) to purified food proteins, and their equivalent food-grade preparations, were related to their effects on reducing bitter receptor activation by EGCG in vitro and their bitter-masking potential in vivo. β-Casein, in particular, and several gelatins, are known as strong binders of EGCG, contrary to β-lactoglobulin. Also in the bitter receptor assay, β-casein showed the strongest effect, with a maximum reduction of hTAS2R39 activation of about 93%. A similar potency was observed for Na-caseinate, which was applied as food-grade alternative for β-casein. β-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. β-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 bitter-masking.

Chapter 6discusses the findings presented in this thesis, addresses prospects and limitations of the bitter receptor cell assay, presents additional results on testing (iso)flavonoids for possible antagonistic properties, and compares taste evaluation by sensory tests, receptor assays and modeling. Furthermore, it evaluates strategies for bitter taste reduction, and applies the findings to soy products and tea.

The systematic investigation of (iso)flavonoid aglycones showed that the substitution pattern of (iso)flavonoids is of higher importance for bitter receptor activation than the backbone structure. In case of bitter receptor antagonists, the substitution pattern as well as backbone structure revealed to be crucial for functionality. The bitter receptor assay was shown to be an appropriate tool not only for identification of bitter receptor agonists and antagonists, but also for identification of reduced receptor activation by complexing agents. Based on the findings of this thesis, it was concluded that complexation with food proteins is the most promising strategy to reduce bitter taste of flavonoids in tea. On the other hand, for soybean isoflavones, debittering by use of bitter receptor blockers seemed to be a promising debittering strategy. Alternatively to the use of receptor blockers, processing conditions (leading to low isoflavone aglycone formation) or raw material choice (i.e. cultivars low in genistein forms) were recommended. In conclusion, the choice of debittering strategies depends on the molecular structure of the bitter food compounds, as exemplified for soybean products and tea. Therefore, each food product seems to require its own tailor-made debittering solution.

Managing technological aspects of Lupinus mutabilis from a food sovereignty perspective in Ecuador
Carvajal Larenas, F.E. - \ 2013
Wageningen University. Promotor(en): Tiny van Boekel, co-promotor(en): Anita Linnemann; Rob Nout. - S.l. : s.n. - ISBN 9789461736789 - 222
lupinus mutabilis - bitterheid - voedselverwerking - voedselgewassen - ecuador - toxiciteit - voedselsoevereiniteit - bitterness - food processing - food crops - toxicity - food sovereignty

The present thesis deals with the technological aspects of the debittering process of lupin in a food sovereignty framework. Of all investigated lupin species,Lupinus mutabilishas the best nutritional composition, which is similar to that of soya bean (Glycine max). Lupins can be used to fortify the protein content of many products. In addition, specific lupin protein concentrates or isolates display functional properties of industrial interest (as emulsifier, gelling and foaming agent). On the other hand, lupins contain bitter alkaloids that have to be removed prior consumption because are toxic (the fatal acute dose of lupin alkaloids was reported as 10 mg kg-1 body weight (bw) for infants and children, and 25 mg kg-1 bw for adults). However, alkaloids also have medical uses for their hypocholesterolemic, anti-arrhythmic and immunosuppressive activity. Bitter lupins have been detoxified by biological, chemical or aqueous processes. However, our research concentrated on the aqueousprocessing because this is the only way to produce debittered lupin for human consumption in an environment-friendly manner.The process investigated involved soaking, cooking, and washing of the raw seeds. This process showed to be inefficient because it took 5.7 ±1.0 days, used water at almost 62 times the weight of the raw dry and bitter lupine, and caused a 22% loss of total solids, principally fat, minerals, and carbohydrates. In addition, the microbiological quality deteriorated during this debittering process. Mathematical modeling based on Fickian diffusion suggested that the diffusion coefficient of alkaloids would be expected to vary between 10-10 and 10-11 m2s-1 because the process is carried out in unsteady conditions. In this scenario, it was clear that a new technology for debittering lupins is needed. For this purpose ahydro-agitator was designed, built and used to test the effect of different washing conditions on alkaloids content, solids in the product, final weight, processing time and water and energy consumption. Results were modeled and optimal processing conditions were inferred from a technological point of view; the optimum solution comprised 18h of soaking, 1h cooking, 3 changes of water/day and 22h of agitation/day. For estimating the washing time a mathematic function was inferred (∂c / ∂t = kc;k= -0.188- 4.973-3*Agitation – 0.0043 * Changes - 1.681-3 Agitation*Changes). Then, the products obtained from different processing conditions were evaluated by consumers on the basis of their willingness to pay in relation to their appreciation scores and product price. Results were modeled. Treatments with more processing increased the product price and diminished liking level. However, the willingness to pay is the combined effect of both variables. For example, people would accept an increase in price of 0.3$/kg if the liking level increases from “like slightly” to “like moderately”. The new developed technology could be used to optimize processes such as hydration and/or removal of undesired materials of legumes and other seeds. The approach used in this study also seems suitable to estimate relationships between processing conditions, liking, price and willingness to pay in other products. The generated information can be helpful in decision making, such as selection of consumers´ preferred process and liking in relation to pricing.

Food proteins as potential carriers for phenolics
Bohin, M.C. - \ 2013
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Jean-Paul Vincken. - S.l. : s.n. - ISBN 9789461736765 - 140
eiwitten - fenolverbindingen - caseïne - caseïnaten - bitterheid - interacties - proteins - phenolic compounds - casein - caseinates - bitterness - interactions

The development of phenolic-rich functional foods is often limited by the off-tastes of phenolics that might be counteracted by sequestering these compounds using a carrier, thereby preventing them to interact with bitter taste receptors and salivary proteins. A range of common animal food proteins were tested for binding of phenolics. It appeared that a proline-rich open protein structure, as in β-casein, favored binding of phenolics. Globular proteins other than bovine serum albumin showed poor potential for use as carrier. No appropriate carriers for monomeric phenolics were found. β-Casein and Na-caseinate were shown to have good bitter-masking potential for EGCG, as measured by a maximal reduction in bitter receptor activation of ~93% measured in vitro. This effective reduction in bitter receptor activation was confirmed by a sensory test. This illustrates the validity of using food proteins with good binding properties as carriers for phenolics.

Different methodologies for probing the interaction between proteins and phenolics were developed: (i) ultrafiltration followed by UV quantification of unbound phenolics in the retentate, (ii) fluorescence quenching, and (iii) ultrafiltration followed by mass spectrometric quantification of unbound phenolics in the retentate. The latter method offered the opportunity to analyze preferential binding to protein of individual phenolics present in a complex mixture. With these methods, it was established that, with respect to phenolics, conformation and flexibility were important drivers of protein-phenolic interaction, besides degree of polymerization and galloylation. With respect to relatively proline-poor unstructured proteins such as α-casein and β-casein, it appeared that there should be other factors, besides proline density, explaining the interaction with phenolics.

A salt reduction of 50% in bread does not decrease bread consumption or increase sodium intake by the choice of sandwich fillings
Bolhuis, D.P. ; Temme, E.H.M. ; Koeman, F. ; Noort, M.W.J. ; Kremer, S. ; Janssen, A.M. - \ 2011
The Journal of Nutrition 141 (2011)12. - ISSN 0022-3166 - p. 2249 - 2255.
dietary-sodium - blood-pressure - taste preference - table salt - flavor - bitterness - mortality - disease - alters
Bread is a major contributor to sodium intake in many countries. Reducing the salt (NaCl) content in bread might be an effective way to reduce overall sodium intake. The objectives of this study were to examine the effects of gradually lowering the salt content in brown bread, with and without flavor compensation (KCI and yeast extract), on bread consumption and sodium intake compensation by choice of sandwich fillings. A total of 116 participants (age: 21 +/- 3 y; BMI: 22 +/- 2 kg/m(2)) consumed a buffet-style breakfast on weekdays for 4 wk. Participants received either regular bread (control group: n = 39), bread whose salt content was gradually lowered each week by 0, 31, 52, and 67% (reduced group: n = 38), or bread whose salt content was also gradually lowered each week but which was also flavor compensated (compensated group: n = 39). A reduction of up to 52% of salt in bread did not lead to lower consumption of bread compared to the control (P = 0.57), whereas less bread was consumed when salt was reduced by 67% (P = 0.006). When bread was flavor compensated, however, a reduction of 67% did not lead to lower consumption (P = 0.69). Salt reduction in bread (with and without flavor compensation) did not induce sodium intake compensation (P = 0.31). In conclusion, a salt reduction of up to 52% in bread or even up to 67% in flavor-compensated bread neither affected bread consumption nor choice of sandwich fillings
PROP sensitivity reflects sensory discrimination between custard desserts
Wijk, R.A. de; Dijksterhuis, G. ; Vereijken, P.H. ; Prinz, J.F. ; Weenen, H. - \ 2007
Food Quality and Preference 18 (2007)4. - ISSN 0950-3293 - p. 597 - 604.
taster status - texture - 6-n-propylthiouracil - perception - bitterness - intensity - ptc/prop - liquid - foods
Sensitivity to 6-n-propylthiouracil (PROP) for a group of 180 naïve consumers was related to their perception of 16 commercially available vanilla custard desserts. Rated intensities of taste and texture attributes varied moderately and inconsistently with PROP sensitivity. In contrast, discriminative ability increased with PROP sensitivity resulting in higher numbers of significant differences between pairs of custards. In terms of signal/noise theory, the results indicate that PROP sensitivity enhances the separation of the response signals but does not reduce their noise. The naïve consumers were also compared with highly trained panelists to test whether effects of PROP sensitivity resemble the effects of experience and training. Naïve consumers and trained panelists responded similarly with respect to taste and texture sensations such as creaminess and thickness, but were clearly different with respect to others such as heterogeneity and fattiness. Trained panelists demonstrated even stronger discriminative abilities than consumers with high PROP sensitivities for some attributes but weaker abilities for others. A practical implication of these findings is that selection criteria for participation in sensory panels should include PROP sensitivity, if the panel is aimed at maximum discriminative performance.
The role of friction in perceived oral texture
Wijk, R.A. de; Prinz, J.F. - \ 2005
Food Quality and Preference 16 (2005)2. - ISSN 0950-3293 - p. 121 - 129.
astringency - perception - bitterness - particles - tannin - wine
Instrumentally measured in vitro friction in semi-solid foods was related to oral texture sensations. Increased fat content resulted in lower sensations of roughness, higher sensations of creaminess, and lower friction, suggesting that lubrication is the mechanism by which fat affects oral texture in low fat foods. Starch breakdown by salivary amylase in low fat foods resulted in reduced friction, possibly through the release of fat from the starch food matrix, and the migration of fat to the surface of the bolus where it becomes available for lubrication. No evidence was found that salivary mucins or salivary viscosity play a role in lubrication. Astringent sensations may be related to reduced lubrication and increased friction caused by particles, either resulting from precipitation of salivary protein rich proteins or from flocculation of dead cells.
Aminopeptidase C of Aspergillus niger is a Novel Phenylalanine Aminopeptidase
Basten, E.J.W. ; Dekker, P.J.T. ; Schaap, P.J. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 1246 - 1250.
flavored peptides - sequence-analysis - gene - cloning - proteins - enzyme - yeast - carboxypeptidase - bitterness - expression
A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine beta-naphthylamide (F-betaNA) and phenylalanine para-nitroanilide (F-betaNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to betaNA; other amino acids with nonaromatic side chains coupled to either pNA or betaNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.
Biochemical and functional characterisation of casein and whey protein hydrolysates : a study on the correlations between biochemical and functional properties using multivariate data analysis
Ven, C. van der - \ 2002
Wageningen University. Promotor(en): A.G.J. Voragen; Harry Gruppen; D.B.A. de Bont. - S.l. : S.n. - ISBN 9789058086532 - 155
melkeiwitten - caseïnehydrolysaat - wei-eiwit - eiwithydrolysaten - peptiden - multivariate analyse - vloeistofchromatografie met omgekeerde fase - gelfiltratiechromatografie - infraroodspectroscopie - molecuulgewicht - emulsies - schuim - oplosbaarheid - bitterheid - milk proteins - casein hydrolysate - whey protein - protein hydrolysates - peptides - multivariate analysis - reverse phase liquid chromatography - gel filtration chromatography - infrared spectroscopy - molecular weight - emulsions - foams - solubility - bitterness
<p>Whey protein and sodium caseinate were hydrolysed with commercially available enzyme preparations. The resulting hydrolysates were characterised using several analytical characterisation methods and by determination of several functional properties. Subsequently, correlations between the biochemical characteristics themselves and between biochemical and functional properties were studied using multivariate regression analysis.</p><p>Biochemical characteristics of hydrolysates were determined using unifactorial methods like the degree of hydrolysis, and by multifactorial methods, <em>i.e</em> . reversed phase (RPC) and size exclusion chromatography (SEC), and Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy appeared to discriminate most effectively between hydrolysates made from different protein sources and classes of proteolytic enzymes, followed by RPC and SEC.</p><p>Emulsion and foam properties of hydrolysates were similar or inferior to those of the parental proteins. Casein hydrolysates generally showed better emulsion and foam forming ability than whey protein hydrolysates. Foam forming ability of whey protein hydrolysates was correlated to the molecular weight distribution (MWD) of the peptides, showing that especially peptides with MW of 3-5 kDa contributed to foam forming ability.</p><p>Concerning prevention of emulsion instability due to coalescence it was shown that peptides with a molecular weight larger than 2 kDa are needed. Foam stabilising ability of casein hydrolysates also depended on the MWD of hydrolysates, but higher molecular weight peptides, <em>i.e.</em> larger than 7 kDa, were needed to obtain good foam stability.</p><p>The ability of the three multifactorial characterisation methods (SEC, RPC, FTIR spectroscopy) to predict functional properties was investigated. It appeared that SEC profiles were able to predict emulsion and foam stability of all hydrolysates, as well as foam forming ability, Angiotensin Converting Enzyme (ACE) inhibiting ability and bitterness of whey hydrolysates. RPC profiles were also able to predict these properties and additionally predicted solubility and bitterness of casein hydrolysates. FTIR spectra were best suited to predict a variety of hydrolysate properties, since apart from the before-mentioned properties, the spectra can also be used to predict emulsion forming ability and to improve prediction of bitterness of hydrolysates.</p><p>Finally, the influence of hydrolysis process conditions on ACE inhibiting ability of whey hydrolysates was investigated, showing that ACE inhibiting activity could be optimised by using process optimisation techniques like experimental design and response surface optimisation.
The biosynthesis of sesquiterpene lactones in chicory (Cichorium intybus L.) roots
Kraker, J.W. de - \ 2002
Wageningen University. Promotor(en): Æ. de Groot; M.C.R. Franssen; Harro Bouwmeester. - S.l. : S.n. - ISBN 9789058085313 - 176
cichorei - biosynthese - sesquiterpenoïde lactonen - secundaire metabolieten - bitterheid - chicory - biosynthesis - sesquiterpenoid lactones - secondary metabolites - bitterness
<FONT FACE="TimesNewRomanPSMT">Wild chicory</font><em><FONT FACE="TimesNewRomanPS-ItalicMT">(Cichorium intybus</em></font><FONT FACE="TimesNewRomanPSMT">L.) is a blue-flowered composite plant that has spread all over the world from the Mediterranean. Sprouts of chicory var.</font><em><FONT FACE="TimesNewRomanPS-ItalicMT">foliosum</em></font><FONT FACE="TimesNewRomanPSMT">Hegi that are grown in the dark became popular as a vegetable (Belgian endive) halfway through the nineteenth century. Nowadays it is a common crop in Belgium, northern France, and the Netherlands. The well-known bitter taste of chicory is associated with the presence of sesquiterpene lactones of which the three major ones are the guaianolides lactucin, 8-deoxylactucin, and lactucopicrin (Fig. 1). Additionally, smaller amounts of eudesmanolides and germacranolides are present. The average sesquiterpene lactone content of the wild variety</font><em><FONT FACE="TimesNewRomanPS-ItalicMT">(sylvestr</em></font><FONT FACE="TimesNewRomanPSMT">e) is 0.42% dry weight in the roots and 0.26% in the leaves. The sesquiterpene lactones in chicory act as feeding deterrent toward insects, but may have an antifungal and antibacterial function as well</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">1). <img src="/wda/abstracts/i3128_1.gif"/></font><br/><br/><FONT FACE="TimesNewRomanPSMT">Sesquiterpene lactones are considered as a major class of plant secondary products, which mainly occur in the Asteraceae. Over 4000 different structures are known, but the majority of them has a guaiane, eudesmane, or germacrene framework (i.e. guaianolides, eudesmanolides, germacranolides). Sesquiterpene lactones with such a framework arethought to originate from (+)-costunolide, the most elementary structure of a germacrene sesquiterpene lactone. In this thesis a pathway for the biosynthesis of (+)-costunolide in chicory roots has been established (Fig. 2). <img src="/wda/abstracts/i3128_2.gif"/></font><br/><br/><strong><FONT FACE="TimesNewRomanPS-BoldMT" SIZE=2>Figure</strong></font><FONT FACE="TimesNewRomanPSMT" SIZE=2>2. Established pathway for the biosynthesis of</font><strong><FONT FACE="TimesNewRomanPS-BoldMT" SIZE=2>(</strong></font><FONT FACE="TimesNewRomanPSMT" SIZE=2>+)-costunolide from farnesyl diphosphate (FPP) in chicory.<br/><br/></font><FONT FACE="TimesNewRomanPSMT">The committed step in the biosynthesis of (+)-costunolide is the cyclisation of farnesyl diphosphate (FPP) into (+)-germacrene A</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">2). The involved (+)-germacrene A synthase was isolated from chicory roots and purified 200-fold by a combination of anion exchange and dye-ligand chromatography. The isolated enzyme belongs to the group of sesquiterpene synthases, has a</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>K</font><FONT FACE="TimesNewRomanPSMT">m-value of 6.6</font><FONT FACE="TT26Do00" SIZE=3>_</font><FONT FACE="TimesNewRomanPSMT">M, an estimated molecular weight of 54 kD, and a (broad) pH optimum around 6.7. The recent isolation of genes encoding the (+)-germacrene A synthase of chicory makes it possibly to block this crucial step in sesquiterpene lactone biosynthesis, which may result in new less bitter tasting varieties of Belgian endive. Formation of the lactone ring involves the introduction of a carboxylic acid function in the isopropenyl group of (+)-germacrene A</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">3). It starts with the hydroxylation of (+)-germacrene A to germacra-1(10),4,11(13)-trien-12-ol by the (+)-germacrene A hydroxylase. This cytochrome P450 enzyme is NADPH-dependent, has a pH optimum at 8.0, and is blue-light reversibly inhibited by CO. Germacra-1(10),4,11(13)-trien-12-ol is subsequently oxidised to germacra-1(10),4,11(13)-trien-12-oic acid via germacra-1( 10),4,11(13)-trien-12-al by pyridine nucleotide dependent dehydrogenases. Some questions about the exact cofactor dependence of the dehydrogenase catalysed reactions remain, but on the whole the best results were obtained with NADP</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>+</font><FONT FACE="TimesNewRomanPSMT">. Conversion of germacra-1(10),4,11(13)-trien-12-oic acid into (+)-costunolide is catalysed by the (+)-costunolide synthase</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">5). This enzyme is also a cytochrome P450 enzyme, since it depends upon NADPH and is blue-light reversibly inhibited by CO. Biosynthesis of (+)-costunolide in the presence of</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>18 O2</font><FONT FACE="TimesNewRomanPSMT">resulted in the incorporation of one atom of</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>18</font><FONT FACE="TimesNewRomanPSMT">O. This supports the concept that the lactone ring is formed via a hydroxylation at the</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>C</font><FONT FACE="TimesNewRomanPSMT">6-position of the germacrene acid, after which the hydroxyl group attacks the carboxyl group at</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>C1</font><FONT FACE="TimesNewRomanPSMT">2. It is not clear whether the final lactonisation is also mediated by the (+)-costunolide synthase or occurs spontaneously (outside the enzyme).</font><FONT FACE="Arial-BoldMT" SIZE=5> <img src="/wda/abstracts/i3128_3.gif"/></font> <br/><FONT FACE="TimesNewRomanPSMT">(+)-Costunolide is converted into 11(S),13-dihydrocostunolide and leucodin by an enzyme extract from chicory roots in the presence of NADPH and</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>O2</font><FONT FACE="TimesNewRomanPSMT">(Fig. 3). It is to be expected that other sesquiterpene lactones are formed as well in these incubations, but it is unlikely thatthey can be detected by the GC-MS method which was used to analyse the enzyme assays. The formation of 11(S),13-dihydrocostunolide is catalysed by a stereoselective enoate reductase and does also occur in the absence of</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>O</font><FONT FACE="TimesNewRomanPSMT">2. The formation of leucodin involves a cytochrome P450 enzyme and presumably also a dehydrogenase, but it is unclear how cyclisation into the guaiane framework takes place. 11(S),13-Dihydrocostunolide is a reasonable intermediate in the biosynthesis of all 11,13-dihydro-sesquiterpene lactones present in chicory, but its involvement in leucodin biosynthesis was not investigated. Notably, leucodin is only one hydroxylation step away from 11(S),13-dihydro-8- deoxylactucin, a minor bitter sesquiterpene lactone of chicory. The germacrene intermediates of sesquiterpene lactone biosynthesis can be isolated from fresh roots of</font><em><FONT FACE="TimesNewRomanPS-ItalicMT">Saussurea lappa</em></font><FONT FACE="TimesNewRomanPSMT">(costus roots)</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">4). The occurrence of these germacrene intermediates along with high amounts of (+)-costunolide and dehydrocostus lactone within one and the same plant is an additional proof for the established pathway depicted in Figure 2. The germacrene compounds are susceptible to proton-induced cyclisations and to heat induced Cope rearrangement yielding eudesmanes and elemenes respectively. However, the isolated germacrenes are not that unstable as often suggested by literature. They remain for instance intact during the enzyme incubations at 30°C. The best way to analyse the oxygenated germacrenes is to let them undergo a Cope rearrangement to their corresponding elemenes immediately at the start of the GC-run. This can be achieved by the use of injection port temperatures of at least 250°C; if this is not done they will generally yield very broad peaks</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">4). Cope rearrangement into (-)-</font><FONT FACE="TT26Do00" SIZE=3>_</font><FONT FACE="TimesNewRomanPSMT">-elemene was used to determine the absolute configuration of the enzymatically produced germacrene A on an enantioselective GC-column</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">2). In the presence of NADPH, a microsomal pellet from chicory roots is able to hydroxylate various sesquiterpene olefins, which are exogenous to the plant</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">6). Most of these hydroxylations take place at the allylic position of an isopropenyl or isopropylidene group (Fig. 4). The number of products obtained from a certain substrate is confined to one or in, a few cases, two sesquiterpene alcohols. Although the microsomal pellet contains various membrane bound enzymes, the majority of hydroxylations is ascribed to the (+)-germacrene A synthase since (+)-germacrene A competitively inhibits their biotransformation. This disputes the common idea that cytochrome P450 enzymes of plant secondary metabolism have a narrow substratespecificity. The unforeseen hydroxylation of (+)-valencene into</font><FONT FACE="TT26Do00" SIZE=3>_</font><FONT FACE="TimesNewRomanPSMT">-nootkatol is presumably catalysed by a different cytochrome P450 enzyme, possibly the same that is involved in biosynthesis of leucodin from (+)-costunolide. During incubation</font><FONT FACE="TT26Do00" SIZE=3>_</font><FONT FACE="TimesNewRomanPSMT">-nootkatol is rapidly oxidised further by NAD(P)</font><FONT FACE="TimesNewRomanPSMT" SIZE=1>+</font><FONT FACE="TimesNewRomanPSMT">-dependent dehydrogenases into nootkatone, a much sought-after component with a distinctive flavour of grapefruit.</font><FONT FACE="ArialMT" SIZE=2><br/></font><br/><img src="/wda/abstracts/i3128_4.gif"/></font><br/><br/><FONT FACE="TimesNewRomanPSMT">The achieved regioselective, and in the case of</font><FONT FACE="TT26Do00" SIZE=3>_</font><FONT FACE="TimesNewRomanPSMT">-nootkatol also stereoselective, introduction of a hydroxyl group into sesquiterpene olefins is often difficult to achieve by organic chemical methods. Nonetheless, the small quantities of oxygenated products obtained are a major drawback in the application of the isolated oxidising enzymes from chicory roots. It would be worthwhile to isolate the genes encoding the involved cytochrome P450 enzymes and to functionally overexpress them in yeast. In this way higher enzymatic activities and larger amounts of possibly interesting products may be expected</font><strong><FONT FACE="TimesNewRomanPS-BoldMT">(Chapter</strong></font><FONT FACE="TimesNewRomanPSMT">7).</font>
Relatie tussen bittere smaak HPLC - profiel van witlof
Mazijk - Bokslag, D.M. van; Cramwinckel, A.B. ; Essers, M.L. ; Hollman, P.C.H. - \ 1991
Wageningen : DLO-Rijks-Kwaliteitsinstituut voor Land- en Tuinbouwprodukten (Rapport / RIKILT-DLO 91.50) - 18
cichorium intybus - cichorei - smaak - bitterheid - hplc - chemische samenstelling - chicory - taste - bitterness - chemical composition
Bitterheid is een belangrijke smaakeigenschap van de witlof. Bitterheid kan als gevolg van raseigenschappen en teeltomstandigheden variëren van bijna niet bitter tot zeer bitter. Op het moment is het voor de consument onduidelijk welke bitterheid hij zal aantreffen. Uit oogpunt van een algemene waardering voor witlof is deze marktsituatie voor verbetering vatbaar. Dit onderzoek wil de mogelijkheden daartoe onderzoeken door eerst het niveau en de spreiding van de bitterheid van verschillende partijen in kaart te brengen. Van enkele partijen witlof zijn sensorische gegevens verzameld. Deze data zijn vergeleken met HPLC profielen ten einde de stoffen te vinden die de bittere smaak geven.
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