Molecular insights into human taste perception and umami tastants: A review.

Understanding taste is key for optimizing the palatability of seaweeds and other non-animal-based foods rich in protein. The lingual papillae in the mouth hold taste buds with taste receptors for the five gustatory taste qualities. Each taste bud contains three distinct cell types, of which Type II cells carry various G protein-coupled receptors that can detect sweet, bitter, or umami tastants, while type III cells detect sour, and likely salty stimuli. Upon ligand binding, receptor-linked intracellular heterotrimeric G proteins initiate a cascade of downstream events which activate the afferent nerve fibers for taste perception in the brain. The taste of amino acids depends on the hydrophobicity, size, charge, isoelectric point, chirality of the alpha carbon, and the functional groups on their side chains. The principal umami ingredient monosodium l-glutamate, broadly known as MSG, loses umami taste upon acetylation, esterification, or methylation, but is able to form flat configurations that bind well to the umami taste receptor. Ribonucleotides such as guanosine monophosphate and inosine monophosphate strongly enhance umami taste when l-glutamate is present. Ribonucleotides bind to the outer section of the venus flytrap domain of the receptor dimer and stabilize the closed conformation. Concentrations of glutamate, aspartate, arginate, and other compounds in food products may enhance saltiness and overall flavor. Umami ingredients may help to reduce the consumption of salts and fats in the general population and increase food consumption in the elderly.

Extraction and analysis of free amino acids and 5'-nucleotides, the key contributors to the umami taste of seaweed.

Assessing the umami taste of seaweed on a chemical level can inform the use and selection of seaweed in European cuisine. Accordingly, we developed a method for the simultaneous extraction, separate clean-up and analysis of 21 free amino acids and 10 free nucleotides by reversed phase and mixed-mode HPLC respectively. Of multiple mouth emulating solvents, extracting in Milli-Q at 35 °C was found most suitable. This method showed good linearity (R2 > 0.9996), resolution (Rs ≥ 1.5) and picomole detection limits, and was successfully applied to determine the Equivalent Umami Concentration (EUC) and Taste Activity Values (TAV) of seven Dutch seaweed species. Phaeophyceae showed the highest EUC, followed by Chlorophyceae and Rhodophyceae (≈ 9.5, 3.7 and 1.1 g/100 g respectively). Glutamic acid always exceeded the TAV, while other umami compounds were species specific. Our method can accurately predict umami intensity and therefore contributes towards species selection for the European palette.

Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates.

RATIONALE: We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ(13)C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although LC/IRMS is expected to be more accurate and precise, no direct comparison has been reported. METHODS: GC/IRMS with the aldonitrile penta-acetate (ANPA) derivatisation method was compared with LC/IRMS without derivatisation. A large number of glucose standards and a variety of natural samples were analysed for five neutral carbohydrates at natural abundance as well as at (13)C-enriched levels. Gas chromatography/chemical ionisation mass spectrometry (GC/CIMS) was applied to check for incomplete derivatisation of the carbohydrate, which would impair the accuracy of the GC/IRMS method. RESULTS: The LC/IRMS technique provided excellent precision (±0.08‰ and ±3.1‰ at natural abundance and enrichment levels, respectively) for the glucose standards and this technique proved to be superior to GC/IRMS (±0.62‰ and ±19.8‰ at natural abundance and enrichment levels, respectively). For GC/IRMS measurements the derivatisation correction and the conversion of carbohydrates into CO2 had a considerable effect on the measured δ(13)C values. However, we did not find any significant differences in the accuracy of the two techniques over the full range of natural δ(13)C abundances and (13)C-labelled glucose. The difference in the performance of GC/IRMS and LC/IRMS diminished when the δ(13)C values were measured in natural samples, because the chromatographic performance and background correction became critical factors, particularly for LC/IRMS. The derivatisation of carbohydrates for the GC/IRMS method was complete. CONCLUSIONS: Although both LC/IRMS and GC/IRMS are reliable techniques for compound-specific stable carbon isotope analysis of carbohydrates (provided that derivatisation is complete and the calibration requirements are met), LC/IRMS is the technique of choice. The reasons for this are the improved precision, simpler sample preparation, and straightforward isotopic calibration.

A versatile method for simultaneous stable carbon isotope analysis of DNA and RNA nucleotides by liquid chromatography/isotope ratio mass spectrometry.

RATIONALE: Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is currently the most accurate and precise technique for the measurement of compound-specific stable carbon isotope ratios ((13)C/(12)C) in biological metabolites, at their natural abundance. However, until now this technique could not be applied for the analysis of nucleic acids, the building blocks of the carriers of genetic information in living cells and viruses, DNA and RNA. METHODS: Mixed-mode chromatography (MMC) was applied to obtain the complete separation of nine nucleotides (eight originating from DNA/RNA and one nucleotide (inosine monophosphate) that may serve as an internal standard) in a single run using LC/IRMS. We also developed and validated a method for DNA and RNA extraction and an enzymatic hydrolysis protocol for natural samples, which is compatible with LC/IRMS analysis as it minimizes the carbon blank. The method was used to measure the concentration and stable carbon isotope ratio of DNA and RNA nucleotides in marine sediment and in the common marine macro alga (Ulva sp.) at natural abundance levels as well as for (13)C-enriched samples. RESULTS: The detection limit of the LC/IRMS method varied between 1.0 nmol for most nucleotides and 2.0 nmol for late-eluting compounds. The intraday and interday reproducibility of nucleotide concentration measurements was better than, respectively, 4.1% and 8.9% and for δ(13)C measurements better than, respectively, 0.3‰ and 0.5‰. The obtained nucleic acid concentrations and nucleic acid synthesis rates were in good agreement with values reported in the literature. CONCLUSIONS: This new method gives reproducible results for the concentration and δ(13)C values of nine nucleotides. This solvent-free chromatographic method may also be used for other purposes, such as for instance to determine nucleotide concentrations using spectrophotometric detection. This sensitive method offers a new avenue for the study of DNA and RNA biosynthesis that can be applied in various fields of research.

Release of dissolved carbohydrates by Emiliania huxleyi and formation of transparent exopolymer particles depend on algal life cycle and bacterial activity.

The coccolithophore Emiliania huxleyi plays a pivotal role in the marine carbon cycle. However, we have only limited understanding of how its life cycle and bacterial interactions affect the production and composition of dissolved extracellular organic carbon and its transfer to the particulate pool. We traced the fate of photosynthetically fixed carbon during phosphate-limited stationary growth of non-axenic, calcifying E. huxleyi batch cultures, and more specifically the transfer of this carbon to bacteria and to dissolved high molecular weight neutral aldoses (HMW NAld) and extracellular particulate carbon. We then compared the dynamics of dissolved carbohydrates and transparent exopolymer particles (TEP) between cultures of non-axenic and axenic diploid E. huxleyi. In addition, we present the first data on extracellular organic carbon in (non-axenic) haploid E. huxleyi cultures. Bacteria enhanced the accumulation of dissolved polysaccharides and altered the composition of dissolved HMW NAld, while they also stimulated the formation of TEP containing high densities of charged polysaccharides in diploid E. huxleyi cultures. In haploid E. huxleyi cultures we found a more pronounced accumulation of dissolved carbohydrates, which had a different NAld composition than the diploid cultures. TEP formation was significantly lower than in the diploid cultures, despite the presence of bacteria. In diploid E. huxleyi cultures, we measured a high level of extracellular release of organic carbon (34-76%), retrieved mainly in the particulate pool instead of the dissolved pool. Enhanced formation of sticky TEP due to bacteria-alga interactions, in concert with the production of coccoliths, suggests that especially diploid E. huxleyi blooms increase the efficiency of export production in the ocean during dissolved phosphate-limited conditions.

The glycemic response does not reflect the in vivo starch digestibility of fiber-rich wheat products in healthy men.

Starchy food products differ in the rate of starch digestion, which can affect their metabolic impact. In this study, we examined how the in vivo starch digestibility is reflected by the glycemic response, because this response is often used to predict starch digestibility. Ten healthy male volunteers [age 21 ± 0.5 y, BMI 23 ± 0.6 kg/m² (mean ± SEM)] participated in a cross-over study, receiving three different meals: pasta with normal wheat bran (PA) and bread with normal (CB) or purple wheat bran (PBB). Purple wheat bran was added in an attempt to decrease the rate of starch digestion. The meals were enriched in ¹³C and the dual isotope technique was applied to calculate the rate of appearance of exogenous glucose (RaE). The ¹³C-isotopic enrichment of glucose in plasma was measured with GC/combustion/isotope ratio MS (IRMS) and liquid chromatography/IRMS. Both IRMS techniques gave similar results. Plasma glucose concentrations [2-h incremental AUC (iAUC)] did not differ between the test meals. The RaE was similar after consumption of CB and PBB, showing that purple wheat bran in bread does not affect in vivo starch digestibility. However, the iAUC of RaE after men consumed PA was less than after they consumed CB (P < 0.0001) despite the similar glucose response. To conclude, the glycemic response does not always reflect the in vivo starch digestibility. This could have implications for intervention studies in which the glycemic response is used to characterize test products.

Human faecal microbiota display variable patterns of glycerol metabolism.

Significant amounts of glycerol reach the colon microbiota daily through the diet and/or by in situ microbial production or release from desquamated epithelial cells. Some gut microorganisms may anaerobically reduce glycerol to 1,3-propanediol (1,3-PDO), with 3-hydroxypropanal as an intermediate. Accumulation of the latter intermediate may result in the formation of reuterin, which is known for its biological activity (e.g. antimicrobial properties). To date, glycerol metabolism in mixed cultures from the human colon has received little attention. Using in vitro batch incubations of faeces from 10 human individuals, we demonstrated that glycerol addition (140 mM) significantly affects the metabolism and composition of the microbial community. About a third of the samples exhibited rapid glycerol conversion, yielding proportionally higher levels of acetate and 1,3-PDO. In contrast, a slower glycerol metabolism resulted in higher levels of propionate. Furthermore, rapid glycerol metabolism correlated with significant shifts in the Lactobacillus-Enterococcus community, which were not observed in slower glycerol-metabolizing samples. As the conversion of glycerol to 1,3-PDO is a highly reducing process, we infer that the glycerol metabolism may act as an effective hydrogen sink. Given the importance of hydrogen-consuming processes in the gut, this work suggests that glycerol may have potential as a tool for modulating fermentation kinetics and profiles in the gastrointestinal tract.

Analysis of [U-13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques.

The use of stable isotope labelled glucose provides insight into glucose metabolism. The 13C-isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques the samples must be derivatized prior to analysis, which makes sample preparation more labour-intensive and increases the uncertainty of the measured isotopic composition. A novel method for the determination of isotopic enrichment of glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been developed. Using this technique, for which hardly any sample preparation is needed, we showed that both the enrichment and the concentration could be measured with very high precision using only 20 microL of plasma. In addition, a comparison with GC/MS and GC/IRMS showed that the best performance was achieved with the LC/IRMS method making it the method of choice for the measurement of 13C-isotopic enrichment in plasma samples.