Benchtop (60 MHz) proton NMR spectroscopy for quantification of 16-O-methylcafestol in lipophilic extracts of ground roast coffee.

We present a method for analysing the lipophilic fraction extracted from ground coffee beans using 60 MHz proton (1H) NMR spectroscopy. In addition to the triglycerides from coffee oil, spectral features are seen from a range of secondary metabolites, such as various diterpenes. We demonstrate quantitation of a peak attributed to one such compound, 16-O-methylcafestol (16-OMC), which is of interest as a coffee species marker. It is present in low concentrations (<<50 mg/kg) in Coffea arabica L. ('Arabica') beans, but in orders of magnitude greater concentrations in other coffees, in particular the other commercially grown species C. canephora Pierre ex A. Froehner (commonly known as 'robusta'). A series of coffee extracts spiked with 16-OMC analytical standard are used to establish a calibration, and to estimate 16-OMC concentrations in a range of different coffees (Arabicas and blends with robustas). To validate the method, values obtained are compared with an analogous quantitation method that uses high field (600 MHz) NMR spectroscopy. •Quantitation of 16-O-methylcafestol in ground roast coffee extracts using benchtop (60 MHz) NMR spectroscopy•Validated by comparison with quantitative high field (600 Mz) NMR spectroscopy•Detection limit is sufficient for discovering adulteration of Arabica coffee with non-Arabica species.

Authentication of beef versus horse meat using 60 MHz 1H NMR spectroscopy.

This work reports a candidate screening protocol to distinguish beef from horse meat based upon comparison of triglyceride signatures obtained by 60 MHz (1)H NMR spectroscopy. Using a simple chloroform-based extraction, we obtained classic low-field triglyceride spectra from typically a 10 min acquisition time. Peak integration was sufficient to differentiate samples of fresh beef (76 extractions) and horse (62 extractions) using Naïve Bayes classification. Principal component analysis gave a two-dimensional "authentic" beef region (p=0.001) against which further spectra could be compared. This model was challenged using a subset of 23 freeze-thawed training samples. The outcomes indicated that storing samples by freezing does not adversely affect the analysis. Of a further collection of extractions from previously unseen samples, 90/91 beef spectra were classified as authentic, and 16/16 horse spectra as non-authentic. We conclude that 60 MHz (1)H NMR represents a feasible high-throughput approach for screening raw meat.

60 MHz 1H NMR spectroscopy for the analysis of edible oils.

We report the first results from a new 60 MHz 1H nuclear magnetic resonance (NMR) bench-top spectrometer, Pulsar, in a study simulating the adulteration of olive oil with hazelnut oil. There were qualitative differences between spectra from the two oil types. A single internal ratio of two isolated groups of peaks could detect hazelnut oil in olive oil at the level of ∼13%w/w, whereas a whole-spectrum chemometric approach brought the limit of detection down to 11.2%w/w for a set of independent test samples. The Pulsar's performance was compared to that of Fourier transform infrared (FTIR) spectroscopy. The Pulsar delivered comparable sensitivity and improved specificity, making it a superior screening tool. We also mapped NMR onto FTIR spectra using a correlation-matrix approach. Interpretation of this heat-map combined with the established annotations of the NMR spectra suggested a hitherto undocumented feature in the IR spectrum at ∼1130 cm-1, attributable to a double-bond vibration.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Electromyographic responses to prescribed mastication.

The aim was to understand between-volunteer differences in Electromyography (EMG) behaviour during chewing. EMG was used to record the electrical activity of the temporal and masseter muscles of volunteers, who carried out mastication movements by operating calibrated springs held between their incisors. The volunteers coordinated their jaw movements with the signal produced by a metronome, at four rates: 30, 60, 90 and 120 beats per minute (bpm). Raw data were analyzed to examine the distributions of the intervals between chews. For the highest prescribed chew rates, the volunteers' distributions were very similar. The distributions varied most for the 30 bpm data, suggesting that volunteers differed in their ability to carry out and maintain this prescribed chewing pattern. The data were Fourier transformed to give power spectra in the frequency domain. The low frequency (<10 Hz) region contained spectral features related to the prescribed chew rate. Principal component analysis of the power spectra revealed that readings from each volunteer clustered together, and the clusters could be largely separated. Such grouping was found irrespective of whether data from each chew rate were analyzed separately or simultaneously. This indicated that within-volunteer variance, arising from the different chew rates as well as between-session variance, is lower than between-volunteer variance; even when individuals are asked to make jaw movements in the same prescribed manner, they can nevertheless be uniquely distinguished by their muscle activity as recorded by EMG.

Cell wall architecture of the elongating maize coleoptile.

The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed.

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

Mid-infrared spectroscopy and chemometrics for the authentication of meat products.

Mid-infrared (MIR) spectroscopy is used to address certain issues connected with the authentication of beef and ox kidney and liver: is it possible to distinguish muscle from offal tissue; does the condition, cut of meat, or type of offal influence the distinction; can pure minced beef be distinguished from that adulterated with offal? Using partial least squares (PLS) and canonical variate analysis, predictive models are developed to identify MIR spectra of beef, kidney, and liver. Using modified SIMCA, the pure beef specimens are modeled as a single class; this model identifies spectra of unadulterated beef as such, with an acceptable error rate, while rejecting spectra of specimens containing 10-100% w/w kidney or liver. Finally, PLS regressions are performed to quantify the amount of added offal. The prediction errors obtained (+/-4.8 and +/-4.0% w/w, respectively, for the kidney and liver calibrations) are commensurate with the detection limits suggested by the SIMCA analysis.

Avoiding overfitting in the analysis of high-dimensional data with artificial neural networks (ANNs).

Complex data analysis is becoming more easily accessible to analytical chemists, including natural computation methods such as artificial neural networks (ANNs). Unfortunately, in many of these methods, inappropriate choices of model parameters can lead to overfitting. This study concerns overfitting issues in the use of ANNs to classify complex, high-dimensional data (where the number of variables far exceeds the number of specimens). We examine whether a parameter ρ, equal to the ratio of the number of observations in the training set to the number of connections in the network, can be used as an indicator to forecast overfitting. Networks possessing different ρ values were trained using as inputs either raw data or scores obtained from principal component analysis (PCA). A primary finding was that different data sets behave very differently. For data sets with either abundant or scant information related to the proposed group structure, overfitting was little influenced by ρ, whereas for intermediate cases some dependence was found, although it was not possible to specify values of ρ which prevented overfitting altogether. The use of a tuning set, to control termination of training and guard against overtraining, did not necessarily prevent overfitting from taking place. However, for data containing scant group-related information, the use of a tuning set reduced the likelihood and magnitude of overfitting, although not eliminating it entirely. For other data sets, little difference in the nature of overfitting arose from the two modes of termination. Small data sets (in terms of number of specimens) were more likely to produce overfit ANNs, as were input layers comprising large numbers of PC scores. Hence, for high-dimensional data, the use of a limited number of PC scores as inputs, a tuning set to prevent overtraining and a test set to detect and guard against overfitting are recommended.

Infrared spectroscopy: instrumental factors affecting the long-term validity of chemometric models.

Data from instrumental techniques such as mid-infrared spectroscopy are increasingly being analyzed for sample identification and classification by chemometric methods based on principal component analysis (PCA). However, even modern spectrometers are subject to instability. This may affect PCA, because PCA selects the variables with the largest variance. This paper investigates the relative effects of sources of instrumental instability using a model developed for fruit puree classification. Single-beam spectra, potentially useful for on-line analysis, saw their overall intensity decrease as the infrared source output and/or the detector sensitivity declined. Consequently, single-beam spectra were mainly differentiated by their overall intensity and had to be used with caution in the long term because this strongly affected the analyses. Absorbance spectra were not sensitive to source or detector decay but showed, in the long term, subtle band shape changes and frequency shifts. While these changes were not found to influence analyses involving very different samples, they diminished the success of analyses of data sets with small intrinsic variance. Where there was large spectral differences between sample classes, instrument-related factors were insignificant. However, where spectral differences were more subtle (with a single class), instrumental effects became more important. Suggestions are given to reduce the instrumental and experimental interferences on chemometric analyses, both when recording spectra and for managing spectral databases.

NMR studies of acetan and the related bacterial polysaccharide, CR1/4, produced by a mutant strain of Acetobacter xylinum.

Acetan is a bacterial polysaccharide produced by Acetobacter xylinum NRRL B42. Chemical mutagenesis of A.xylinum allowed selection of a mutant strain which produced a new polysaccharide, CR1/4. 2D NMR methods have been used to assign the 1H and 13C spectra of the two polysaccharides and to determine that CR1/4 has the structure shown below. The total number of O-acetyl groups is slightly less than two per repeating unit. [formula: see text] The pentasaccharide side chain of acetan is truncated to a disaccharide unit in CR1/4, but the structures are otherwise identical. In particular, the degree of acetylation is about the same and the O-acetyl groups are located at the same position in both polysaccharides.