Robert S. T. Linforth

Atmospheric pressure chemical ionisation mass spectrometry for in vivo analysis of volatile flavour release

Abstract To follow volatile flavour release in the expired air of people during eating, several physiological and analytical constraints must be observed to obtain good quality data. An interface has been developed to sample air from the nose and ionise the volatile compounds contained therein by atmospheric pressure chemical ionisation. The ions formed are detected in a quadrupole mass spectrometer. The interface design overcomes many of the constraints and allows volatile detection in the 100–10 ppbv range, depending on the nature of the volatile compound to be analysed. The principles and operating parameters of the interface are described. Control of the ionisation process is effected through the operating conditions within the interface. Since many compounds are introduced simultaneously into the interface, it is preferable to minimise fragmentation otherwise the spectra are extremely complex and it is difficult to assign ions to compounds unequivocally, with the result that quantification is imprecise. The ionisation parameters are set to favour formation of the protonated molecular (MH + ) ion from the compounds and minimise formation of cluster ions, which can also confuse the spectra. The sensitivity and linearity of the technique is demonstrated for a range of flavour volatiles with a dynamic range of three orders of magnitude. The lower limit of sensitivity is determined by the signal to noise ratio whereas the upper limit occurs when all the available charge is exhausted. The technique was designed primarily to monitor real-time changes in the concentration of known volatiles during eating and has limited identification power. However, for reliable quantification, it is necessary to assign the ions monitored in-nose with the volatile compounds present in the food. Besides assignments made on nominal mass, isotopic ratio analysis and accurate mass measurements have also been used to determine the contribution of certain compounds to a particular ion intensity.

Flavour release in the mouth

Abstract Eating is the stage at which food flavour is released, sensed and judged by consumers. The relationships between flavour compounds and sensory perception are still not entirely clear. Analysing the total flavour composition of a food does not reflect the flavour profile experienced during eating; however, new methods of analysis that model flavour release in-mouth have been developed. Recently, direct analysis of volatiles in the air expired through the nose and mouth has been achieved during eating, confirming that the volatile profile changes over time, as had been suspected from results produced by time-intensity sensory analysis. Data from these analyses may explain the link between perception and food composition as well as providing a tool for optimizing the formulation of flavours in low-fat foods.

Measurement of volatile release in the mouth

Abstract Using extra strong mints as a simple food system, which contains high levels of volatiles and a simple aroma profile, different methods of sampling the volatiles in the mouth during eating have been investigated. Release of volatiles in the mouth is important in determining the profile perceived by the receptors in the nose and thus relates directly to our perception of aroma when food is eaten. Direct introduction of volatiles from the mouth and nose into a mass spectrometer did not provide information on the volatile profile, as the air introduced greatly reduced the sensitivity of the machine, and volatiles could not be reliably detected above the background noise. Cryogenic trapping on fused silica capillaries followed by gas chromatography and mass spectrometry did give profiles which were different for headspace, mouthspace and nosespace. Problems with loss of volatiles after trapping and the presence of water on the traps limited the usefulness of this method. Trapping on Tenax traps overcame some of these problems, and similar profiles to those obtained with cryogenic trapping were obtained. The amounts of two major volatiles in the headspace and nosespace were estimated and found to be menthone (8·32 and 24·3 mg m 3 air) and menthol (2·59 and 4·3 mg m 3 air), respectively. The concentrations of menthone in both headspace and nosespace were below the reported odour threshold value, but menthol was present in concentrations above the odour threshold. The method shows that volatile profiles during eating can be measured, but further development is required to improve sensitivity if the technique is to be applied to other foods.

Perception of taste intensity in solutions of random-coil polysaccharides above and below c∗

Sensory paired comparison tests were used to study differences in taste intensity in solutions of hydroxypropylmethyl cellulose (HPMC) at concentrations above (1.0% w/w) and below (0.2% w/w) c*, the coil-overlap concentration (the point at which viscosity changes abruptly with increasing thickener). The sweetness intensities of aspartame (250 ppm), sucrose (5% w/w), fructose (4.5% w/w) and neohesperidin dihydrochalcone (39 ppm) and the saltiness of sodium chloride (0.35%) were all found to be significantly reduced in the more viscous HPMC solution. There was no significant effect of HPMC concentration on the acidity of citric acid (600 ppm) or the bitterness of quinine hydrochloride (26 ppm). The sweetness intensities of sucrose and aspartame were likewise investigated in two further hydrocolloid solutions, guar gum and λ-carrageenan. Experiments were designed so that the ratios of the thickener concentrations (above and below c*) to their measured c* values remained constant. The sweetness of sucrose was found to be significantly reduced in the more viscous guar gum solution (P<0.05) and that of aspartame was reduced in the λ-carrageenan above c* (P<0.001). A multiple paired comparison design was used to show that the perceived sweetness of 6.5% sucrose in 1.0% HPMC did not differ significantly from that of 5% sucrose in 0.2% HPMC. The magnitude of effect with aspartame was broadly analogous.

Photocatalytic CO2 reduction using an internally illuminated monolith photoreactor

One of the promising solutions to both global climate warming and increasing energy demands is artificial photosynthesis, which can be implemented via the photoreduction of CO2 to produce fuel. A monolith photoreactor was used to increase the amount of catalyst loading due to its multiple channels. The photocatalyst was dip coated using NiO/InTaO4 sol and then calcined at 1100 °C. A uniform NiO/InTaO4 layer was obtained on the top of pre-coated SiO2 sublayer on the internal channels of the monolith. The polymethylmethacrylate (PMMA) optical fibers, after being carved on their surface, could transmit and scatter light to effectively illuminate the catalyst inside the channels of the monolith. Vapor-phase CO2 with H2O was photocatalytically reduced to hydrocarbons by UV or visible-light in a steady-state flow mode. The maximum methanol conversion rate achieved was 0.16 µmol g−1 h−1 with visible-light of 290 klx at 25 °C. The highest rate of acetaldehyde was 0.3 µmol g−1 h−1 which was obtained with a loading of 2.6% NiO by simulated sunlight AM1.5G at 70 °C. More importantly, the quantum efficiency was significantly improved indicating that photon energy was effectively utilized in the monolith reactor, compared with previous optical-fiber reactor.

An empirical model to predict the headspace concentration of volatile compounds above solutions containing sucrose.

Abstract An empirical model was developed to describe and predict the change in gas–liquid partition behaviour of a wide range of volatile compounds in aqueous sucrose solutions. The static equilibrium headspace concentrations of 40 volatiles (from different chemical classes e.g. pyrazines, alcohols, esters and ketones and with different physical properties e.g. volatility and solubility), were measured above aqueous sucrose solutions [0–65% (w/v)]. As sugar concentration increased, the headspace concentration of some compounds increased, whilst others stayed the same or decreased. The changes in volatile headspace concentrations were correlated (using partial least squares regression) with a range of physicochemical descriptors which were calculated from the compound structure. The model was composed of seven physicochemical descriptors and had a regression coefficient, R 2 =0.74. An external test set was used to validate the model. The key descriptors in the model were (log P ) 2 , LUMO energy and a connectivity index term.

Fruit-specific lipoxygenase suppression in antisense-transgenic tomatoes

Abstract To determine the importance of the enzyme lipoxygenase (LOX) in the generation of volatile C6 aldehyde and alcohol flavour compounds, two antisense LOX genes were constructed and transferred to tomato plants. The first of these constructs (p2ALX) incorporated the fruit-specific 2A11 promoter [Van Haaren, M.J.J., Houck, C.M., 1993. A functional map of the fruit-specific promoter of the tomato 2A11 gene. Plant Mol. Biol. 21, 625–640] and a 1.2 Kb antisense fragment of the cDNA of PTL1 (TomloxA) [Ferrie, B.J., Beaudoin, N., Burkhart, W., Bowsher, C.G., Rothstein, S.J., 1994. The cloning of two tomato lipoxygenase genes and their differential expression during tomato fruit ripening. Plant Physiol. 106, 109–118]; no terminator was included. The second construct (pPGLX) consisted of the ripening-specific polygalacturonase (PG) promoter and terminator [Nicholass, F.J., Smith, C.J.S., Schuch, W., Bird, C.R., Grierson, D., 1995. High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions. Plant Mol. Biol. 28, 423–435] and a 400 bp antisense fragment of the tomato cDNA of PTL1 (TomloxA). Both constructs included the highly conserved LOX region (approximately 110 bp) shared by all plant and mammalian lipoxygenase genes. Reduced levels of endogenous TomloxA and TomloxB mRNA (2–20% of wild-type) were detected in transgenic fruit containing the p2ALX construct compared to non-transformed plants, whereas the levels of mRNA for a distinct isoform, TomloxC, were either unaffected or even increased. The pPGLX construct was much less effective in reducing endogenous LOX mRNA levels. In the case of the p2ALX plants, LOX enzyme activity was also greatly reduced compared with wild-type plants. Analysis of flavour volatiles, however, indicated that there were no significant changes. These findings suggest that either very low levels of LOX are sufficient for the generation of C6 aldehydes and alcohols, or that a specific isoform such as TomloxC, in the absence of TomloxA and TomloxB, is responsible for the production of these compounds.

Volatile production in tomato fruit with modified alcohol dehydrogenase activity

Using a low shear maceration procedure, headspace volatiles were collected and analysed from tomato fruit which had been genetically modified to enhance or reduce the activity of alcohol dehydrogenase 2 (ADH2). Fruit with enhanced activity showed up to three times the activity of control plants while down-regulated fruit showed about 3% of the control ADH activity. The aldehyde to alcohol ratios for (Z )-3-hexenal : (Z)-3-hexenol and 3-methylbutanal : 3-methylbutanol were calculated and shown to differ for down-regulated fruit compared to control fruit. Up-regulation showed no significant increase in either ratio. The amounts of (Z)-3-hexenol or 3-methylbutanol were not significantly increased by up-regulation. Down-regulation caused a significant decrease in the amount of (Z )-3-hexenol but no increase in (Z )-3-hexenal. In contrast, the amount of 3-methylbutanal increased while the amount of 3-methylbutanol did not change significantly. Since 3-methylbutanal and its alcohol are formed over the whole ripening period while (Z)-3-hexenal is formed only in the short period when tomato fruit are macerated, a potential explanation for these differences can be proposed. © 1999 Society of Chemical Industry

Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5),11(12)-diene] in transgenic tomato fruit

The taxanes are a group of polycyclic diterpenes produced by various species of yew. The potent anticancer drug paclitaxel (marketed as Taxol™) is the commercially most important taxane with annual sales in 2000 exceeding

Simultaneous instrumental and sensory analysis of volatile release from gelatine and pectin/gelatine gels

1.6 billion. Paclitaxel is currently obtained either by direct extraction from yew trees or by the extraction of the precursor 10-deactilbaccatin III, which is then converted to paclitaxel by semi-synthesis. Apart from cost, one of the main draw backs to taxol in cancer treatment is the development of resistance by tumours, commonly due to the expression of ABC transporter efflux pumps which remove the drug from the target cell. A number of natural taxanes and semisynthetic derivates, have recently been shown to act as potent inhibitors of ABC transport proteins. These compounds have no effect upon microtubule polymerization (the normal target of paclitaxel), but have the ability to restore drug sensitivity when given in combination with paclitaxel to resistant cell lines. In work to be described elsewhere, we sort to carry out a structure function analysis of the ability of novel oxidised taxanes to act as ABC transporter inhibitors. For this study 100 mg or more of taxadiene [taxa-4(5),11(12)-diene], the first taxane in the paclitaxel pathway, was required as starting material from which to synthesize these compounds. Taxadiene is synthesised directly from geranylgeranyl diphosphate (GGPP), which is found in most plant tissues where it serves as a common precursor for many metabolites. The synthesis and use of GGDP are tightly regulated in most vegetative organs, however, in tomato fruit it is used almost exclusively for the production of coloured carotenoids which accumulate to high levels in the plastid as lycopene crystals. Expressing taxadiene synthase in a yellow-fruited tomato line that lacks the ability to utilise GGPP for carotenoid synthesis allowed GGPP normally utilised for making carotenoids to be re-routed for the production of taxadiene, allowing the facile extraction of 160 mg of highly pure taxadiene from 1 kg of freeze dried fruit.