Luciano Navarini

Polysaccharides from hot water extracts of roasted Coffea arabica beans: isolation and characterization

Abstract A polysaccharide fraction was obtained from the hot water extract of defatted ground dark roasted coffee (pure Coffea arabica blend for espresso brewing technique) by means of classical precipitation methods. This high molecular weight (〈 M w 〉=10u2008900xa0Da) product was shown to be composed of mannose, galactose, arabinose and traces of proteinaceous material. Further fractionation yielded two different carbohydrate polymers, which were structurally characterized. One polysaccharide was identified as a β - d -(1-4) mannan containing small amounts of galactose and arabinose. The second polysaccharide, obtained in low yield after removal of the mannose-containing polymeric material, was identified as an arabinogalactan. The starting fraction was found to be composed of about 80% (mol basis) of mannan and of about 20% of arabinogalactan. 13 C-NMR spectra revealed that the arabinogalactan has a backbone chain of β -(1-3)- d -galactopyranose units. Some of these units were substituted with either terminal β - d -galactose or terminal α - l- arabinofuranose side chains mainly in C-6 position. Both the mannan and the arabinogalactan isolated are structurally related to the polysaccharides originally present in the green coffee beans.

Isolation and characterization of the exopolysaccharide produced by Streptococcus thermophilus SFi20.

This paper reports isolation, structural characterization and some physico-chemical properties in aqueous solution of the exopolysaccharide (EPS) produced by Streptococcus thermophilus strain SFi20. The yield of the purified EPS was found to be reproducible and close to the average value of 143 mg/l. The chemical structure, previously suggested, has been confirmed on the basis of NMR data. Viscometric, chiro-optical and rheological measurements have been carried out with the aim of characterizing the conformational state of the polysaccharide in aqueous solution. All the data reported indicate that the EPS does not undergo a cooperative conformational transition under the investigated experimental conditions. Furthermore, the viscosity data and the viscoelastic behaviour suggest that the polymer is rather flexible and adopts a random coil conformation in aqueous solution.

Interaction of chlorogenic acids and quinides from coffee with human serum albumin.

Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-γ-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-γ-quinide), finding dissociation constants of the albumin-chlorogenic acids and albumin-quinides complexes in the micromolar range, between 2 and 30μM. Such values are comparable with those of the most powerful binders of albumin, and more favourable than the values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5-γ-quinide, we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dissociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by the quinide has also been demonstrated.

Production of bacterial exopolysaccharides by solid substrate fermentation

Abstract In a comparison of submerged cultivation (SC) with solid substrate fermentation (SSF) for the production of bacterial exopolysaccharides (EPS), the latter technique yielded 2 to 4.7 times more polymer than the former, on the laboratory scale. SSF was performed using inert solid particles (spent malt grains) impregnated with a liquid medium. The polymer yields obtained from SSFs, as referred to the impregnating liquid volumes, were as follows: 38.8 g/litre xanthan from Xanthomonas campestris , 21.8 g/litre succinoglycan from Rhizobium hedysari and 20.3 g/litre succinoglycan from Agrobacterium tumefaciens PT45. These results make this technique promising for a potential application on the industrial scale. A further advantage with this fermentation process is found in the availability and low cost of substrates, which are obtained as by-products or wastes from the agriculture or food industry.

Rapid authentication of coffee blends and quantification of 16-O-methylcafestol in roasted coffee beans by nuclear magnetic resonance.

Roasted coffee is subject to commercial frauds, because the high-quality Coffea arabica species, described as 100% Arabica or Highland coffee, is often mixed with the less expensive Coffea canephora var. Robusta. The quantification of 16-O-methylcafestol (16-OMC) is useful to monitor the authenticity of the products as well as the Robusta content in blends. The German standard method DIN 10779 is used in the determination of 16-OMC in roasted coffee beans to detect C. canephora in blends, but it is laborious and time-consuming. Here, we introduce a new method that provides a quantitative determination of esterified 16-OMC directly in coffee extracts by means of high-resolution proton nuclear magnetic resonance spectroscopy. Limit of detection and limit of quantitation were 5 and 20 mg/kg, respectively, which are adequate to detect the presence of Robusta at percentages lower than 0.9%. The proposed method is much faster, more sensitive, and much more reproducible than the DIN standard method.

Green coffee oil analysis by high-resolution nuclear magnetic resonance spectroscopy

In this work, we show how an extensive and fast quantification of the main components in green coffee oil can be achieved by NMR, with minimal sample manipulation and use of organic solvents. The approach is based on the integration of characteristic NMR signals, selected because of their similar relaxation properties and because they fall in similar spectral regions, which minimizes offset effects. Quantification of glycerides, together with their fatty acid components (oleic, linoleic, linolenic and saturated) and minor species (caffeine, cafestol, kahweol and 16-O-methylcafestol), is achieved in less than 1h making use of (1)H and (13)C spectroscopy. The compositional data obtained are in reasonable agreement with classical chromatographic analyses.

Interaction of coffee compounds with serum albumins. Part II: Diterpenes

Cafestol and 16-O-methylcafestol are diterpenes present in coffee, but whilst cafestol is found in both Coffea canephora and Coffea arabica, 16-O-methylcafestol (16-OMC) was reported to be specific of only C. canephora. The interactions of such compounds, with serum albumins, have been studied. Three albumins have been considered, namely human serum albumin (HSA), fatty acid free HSA (ffHSA) and bovine serum albumin (BSA). The proteins interact with the diterpenes at the interface between Sudlow site I and the fatty acid binding site 6 in a very peculiar way, leading to a significant change in the secondary structure. The diterpenes do not displace reference binding drugs of site 2, but rather they enhance the affinity of the site for the drugs. They, therefore, may alter the pharmacokinetic profile of albumin - bound drugs.

Structural characterization and solution properties of an acidic branched (1→3)-β-D-glucan from Aureobasidium pullulans☆

An acidic exopolysaccharide was isolated from a selected strain of Aureobasidium pullulans. On the basis of spectroscopic and chromatographic techniques, the polymer was identified as a beta-D-glucan containing a main chain of (1-->3)-linked beta-D-glucopy-ranosyl units substituted at the O-6 position by single beta-D-glucopyranosyl side chains. The ratio of units in the main chain to units in the side chain was found to be 1.4:1. The ionic character of this exopolysaccharide is due to the presence of malate residues which are linked to the polymer through ester bonds. The degree of substitution was estimated to be very low (0.05). In aqueous solution no signals are present in the NMR spectra strongly suggesting that the polymer adopts a rigid ordered conformation as further confirmed by rheological data. A solvent-induced conformational transition was observed in DMSO in which NMR spectra with good signal-to-noise ratio were obtained. The solution behaviour of the polymer is similar to that of other branched (1-->3)-beta-D-glucans in spite of both the degree of branching and the substitution with malate groups.

Preliminary Characterization of Monofloral Coffea spp. Honey: Correlation between Potential Biomarkers and Pollen Content

To determine the botanical origin of Coffea honey, a new method using proton nuclear magnetic resonance ((1)H NMR) is proposed. Integration of the aromatic region of the NMR spectrum of Coffea honey diluted in deuterated water allowed us to simultaneously quantify caffeine, theobromine, and trigonelline, as well as other compounds. The amounts of the three markers listed are significantly higher than those previously reported for Citrus spp. honey: caffeine ranged from 15 to 98 mg/kg, theobromine from 25 to 160 mg/kg, and trigonelline from 23 to 86 mg/kg. The concurrent presence of these three substances is proposed as an indicator of the botanical origin of Coffea honey. Excellent correlation was found between these markers and the relative amounts of Coffea pollen measured in the same samples.

Quantification of caffeine in human saliva by Nuclear Magnetic Resonance as an alternative method for cytochrome CYP1A2 phenotyping.

The first step in caffeine metabolism is mediated for over 95% by the CYP1A2 isoform of cytochrome P450. Therefore, CYP1A2 activity is most conveniently measured through the determination of caffeine clearance. The HPLC quantification of caffeine is fully validated and is the most widely used method. It can be performed on saliva, which is gaining importance as a diagnostic biofluid and permits easy and low invasive sampling. Here, we present a quantitative (1)H nuclear magnetic resonance (NMR) method to determine caffeine in human saliva. The procedure is simple because it involves only an ultra-filtration step and a direct extraction in a deuterated solvent, yielding a matrix that is then analyzed. The reliability of this NMR method was demonstrated in terms of linearity, accuracy, recovery, and limits of detection (LoD). Good precision (relative standard deviation, RSD <4%), a recovery of >95% and LoD of 6.8·10(-7) mol L(-1) were obtained. The method was applied to samples collected from different volunteers over 24h following a single oral dose of about 100mg of caffeine administered with either coffee beverage or a capsule.