Use of NMR-Based Metabolomics To Chemically Characterize the Roasting Process of Chicory Root

Roasted chicory root (Cichorium intybus) has been widely accepted as the most important coffee substitute. In this study, a nuclear magnetic resonance (NMR)-based comprehensive analysis was performed to monitor the substantial changes in the composition of chicory root during the roasting process. A detailed signal assignment of dried raw and roasted chicory roots was carried out using 1H, 13C, 1H–1H DQF-COSY, 1H–13C edited-HSQC, 1H–13C CT-HMBC, and 1H–13C HSQC-TOCSY NMR spectra. On the basis of the signal assignments, 36 NMR-visible components were monitored simultaneously during roasting. Inulins, sucrose, and most of the amino acids were largely degraded during the roasting process, whereas monosaccharides decreased at the beginning and then increased until the dark roasting stage. Acetamide, 5-hydroxymethylfurfural, di-d-fructose dianhydride, and norfuraneol were newly formed during roasting. Furthermore, a principal component analysis score plot indicated that similar chemical composition profiles could be achieved by roasting the chicory root either at a higher firepower for a shorter time or at a lower firepower for a longer time

Comparison of Peanut Compounds during Roasting and the Effect of Peanut Shells

Peanuts are widely used in a variety of processed foods, and their food properties are attributed to a variety of compounds that change in the roasting process. Here, we investigated the thermal changes in multiple categories of compounds in Virginia-type peanut seeds and peels during roasting using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–tandem mass spectrometry (LC–MS/MS) as nontargeted analytical methods. Detectable taste and aroma compounds increased up to 2.1- (sucrose), 2.0- (glutamate + pyroglutamate), and 68-fold (4-methyl-5-thiazoleethanol) during roasting. Thermal changes characterizing the functional benefits of peanuts are evident, especially an increase in nicotinate (up to 15-fold) and flavonoids (up to 3.9-fold). Furthermore, the effect of the peanut shell on heat was shown by the different patterns of roasting changes in several compounds, including pyroglutamate, and the changes in amino acids and sugars related to the process of the Maillard reaction were more pronounced

Two-Dimensional ¹H–¹³C Nuclear Magnetic Resonance (NMR)-Based Comprehensive Analysis of Roasted Coffee Bean Extract

Coffee was characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. To identify the coffee components, a detailed and approximately 90% signal assignment was carried out using various two-dimensional NMR spectra and a spiking method, in which authentic compounds were added to the roasted coffee bean extract (RCBE) sample. A total of 24 coffee components, including 5 polysaccharide units, 3 stereoisomers of chlorogenic acids, and 2 stereoisomers of quinic acids, were identified with the NMR spectra of RCBE. On the basis of the signal assignment, state analyses were further launched for the metal ion–citrate complexes and caffeine–chlorogenate complexes. On the basis of the signal integration, the coffee components were successfully quantified. This NMR methodology yielded detailed information on RCBE using only a single observation and provides a systemic approach for the analysis of other complex mixtures

Model of TRBP and PACT binding to siRNA.

<p>(A, B) TRBP-WT (A) or TRBP-ΔdsRBD3 (B) binds one molecule of siRNA as a monomer at low concentrations, and then each protein dimerizes due to the increased protein concentration. However, excessive amount of siRNAs were added, TRBP-WT or TRBP-ΔdsRBD3 dimer was dissociated into monomeric TRBP-WT or TRBP-ΔdsRBD3 dimer containing a single molecule of siRNA. (C) PACT-WT forms homodimers at high concentrations and binds to one or two molecules of siRNA. (D) PACT-ΔdsRBD3 binds one siRNA molecule as a monomer or binds one or two siRNA molecules as a dimer. The monomer and dimer may achieve equilibrium, although the monomeric form is predominant. In C and D, we could not determine whether the siRNA shown in gray is contained in the dimerized PACT proteins or not.</p

Distinguishable <i>In Vitro</i> Binding Mode of Monomeric TRBP and Dimeric PACT with siRNA

<div><p>RNA interference (RNAi) is an evolutionally conserved posttranscriptional gene-silencing mechanism whereby small interfering RNA (siRNA) triggers sequence-specific cleavage of its cognate mRNA. Dicer, Argonaute (Ago), and either TAR-RNA binding protein (TRBP) or a protein activator of PKR (PACT) are the primary components of the RNAi pathway, and they comprise the core of a complex termed the RNA-induced silencing complex (RISC)-loading complex (RLC). TRBP and PACT share similar structural features including three dsRNA binding domains (dsRBDs), and a complex containing Dicer and either TRBP or PACT is considered to sense thermodynamic asymmetry of siRNA ends for guide strand selection. Thus, both TRBP and PACT are thought to participate in the RNAi pathway in an indistinguishable manner, but the differences in siRNA binding mode and the functional involvement of TRBP and PACT are poorly understood. Here, we show <i>in vitro</i> binding patterns of human TRBP and PACT to siRNA using electrophoresis mobility shift analysis and gel filtration chromatography. Our results clearly showed that TRBP and PACT have distinct <i>in vitro</i> siRNA binding patterns from each other. The results suggest that monomeric TRBP binds to siRNA at the higher affinity compared to the affinity for own homodimerization. In contrast, the affinity between PACT and siRNA is lower than that of homodimerization or that between TRBP and siRNA. Thus, siRNA may be more readily incorporated into RLC, interacting with TRBP (instead of PACT) <i>in vivo</i>.</p></div

Roasting Process of Coffee Beans as Studied by Nuclear Magnetic Resonance: Time Course of Changes in Composition

In this paper, we report a ¹H and ¹³C nuclear magnetic resonance (NMR)-based comprehensive analysis of coffee bean extracts of different degrees of roast. The roasting process of coffee bean extracts was chemically characterized using detailed signal assignment information coupled with multivariate data analysis. A total of 30 NMR-visible components of coffee bean extracts were monitored simultaneously as a function of the roasting duration. During roasting, components such as sucrose and chlorogenic acids were degraded and components such as quinic acids, <i>N</i>-methylpyridinium, and water-soluble polysaccharides were formed. Caffeine and <i>myo</i>-inositol were relatively thermally stable. Multivariate data analysis indicated that some components such as sucrose, chlorogenic acids, quinic acids, and polysaccharides could serve as chemical markers during coffee bean roasting. The present composition-based quality analysis provides an excellent holistic method and suggests useful chemical markers to control and characterize the coffee-roasting process

Gel filtration chromatography of purified PACT-WT and PACT mutant proteins with siRNA.

<p>Gel filtration chromatography patterns of non-labeled siLuc-36 (300 nM) with PACT-WT (A), PACT- dsRBDmt1 (B), PACT-dsRBDmt2 (C), and PACT-ΔdsRBD3 (D). Lower panels showed the results of Western blot (WB) by anti-myc antibody for detecting PACT proteins in the elution fractions. Histograms below WBs showed the quantified signal densities of PACT proteins detected by WBs. Arrowheads indicate the positions of molecular weight size markers.</p

Purification of PACT-WT and its mutant proteins, and EMSA of PACT-WT protein.

<p>(A) Coomassie brilliant blue stained pattern of purified PACT-WT (left panel), and its mutant proteins (right panel) resolved by SDS-PAGE. Arrows indicate the migration position of PACT-WT, PACT-dsRBDmt1, PACT-dsRBDmt2, and PACT-dsRBDmt1+2 proteins. Arrowhead indicates PACT-ΔdsRBD3 protein. M represents the protein size markers. (B–D) EMSA patterns of PACT-WT with ³²P-labeled siLuc-36 (B), siLuc-36B (C), and siLuc-36D (D). ³²P-labeled siRNA (0.50 nM) was incubated with increasing amounts of PACT-WT as indicated. Lower cases in D indicate DNAs.</p

Gel filtration chromatography of purified TRBP-WT and TRBP-ΔdsRBD3 proteins with siRNA.

<p>Gel filtration chromatography patterns of non-labeled 300 nM of siLuc-36 alone (A), siLuc-36 with 1,300 nM of TRBP-WT (B), siLuc-36 with 5.1 nM of TRBP-WT(C) and siLuc-36 with 1,300 nM of TRBP-ΔdsRBD3 (D). In A, inset, siLuc-36 was electrophoresed on a 3% agarose gel and stained with EtBr. Lower panels in B-D showed the results of Western blot (WB) by anti-myc antibody for detecting TRBP proteins in the elution fractions. Histograms below WBs showed the quantified signal densities of TRBP proteins detected by WB. Arrowheads indicate the positions of molecular weight size markers. In B, each fraction of molecular weight size markers was quantified and represented in histogram after staining the gel with Coomassie brilliant blue.</p

Purification and EMSA of TRBP-WT protein.

<p>(A) Coomassie brilliant blue stained pattern of purified TRBP-WT protein resolved by SDS-PAGE. Arrowhead indicates 44 kDa of TRBP-WT protein. M represents the protein size markers. (B–D) The results of EMSA of TRBP-WT protein with ³²P-labeled siLuc-36 (B), siLuc-36B (C), and siLuc-36D (D). ³²P-labeled siRNA (0.50 nM) was incubated with increasing amounts of TRBP-WT protein, as indicated. Lower cases in siLuc-36D sequence in D indicate DNAs. (E) Supershift analysis of TRBP-WT protein (1.3 nM) with no antibodies, control anti-Flag, and anti-myc antibodies. (F) The result of EMSA of TRBP-WT protein (1,300 nM) mixed with ³²P-labeled siLuc-36 (0.50 nM) incubated with increasing amount of non-labeled siLuc-36. In B–F, arrows indicate positions of the first and second step migrating complexes, corresponding to TRBP-WT complexes 1 and 2, respectively, and the supershifted complex, in addition to siRNA and ATP.</p