Manuel A. Coimbra

Structure-function relationships of immunostimulatory polysaccharides: A review.

Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity.

FTIR SPECTROSCOPY AS A TOOL FOR THE ANALYSIS OF OLIVE PULP CELL-WALL POLYSACCHARIDE EXTRACTS

Abstract The sequential extraction of olive pulp cell-wall material (CWM) and its subsequent fractionation by ethanol precipitation and anion-exchange chromatography gave a wide range of cell-wall polysaccharide fractions, characterised by sugar analysis. Several multivariate procedures, such as principal component analysis (PCA), trimmed object projections (TOP), canonical correlation analysis (CCA), and partial least squares regression (PLS), were applied to their FTIR spectra, in the region between 1200 and 850 cm−1. The combination of these chemometric techniques along with the chemical information allowed the type of polymers present to be distinguished: pectic polysaccharides rich in uronic acid, pectic polysaccharides rich in arabinose, arabinose-rich glycoproteins, xyloglucans, and glucuronoxylans. It was also possible to highlight the most important and characteristic wavenumbers for each type of polymer present. A calibration model for the quantification of xylose residues of the hemicellulosic polysaccharides was proposed. The high diversity of samples used and their characteristic features allowed models to be obtained, using a very expeditious methodology, that represent a realistic description of the olive pulp cell-wall polymers.

Isolation and Analysis of Cell Wall Polymers from Olive Pulp

The olive tree (Olea europaeia) is native to countries having a Mediterranean climate (Fernandez-Diez 1971, 1983). Olive fruit is a drupe, i.e. a meaty stone fruit (Rommani and Jennings 1971), and is usually an elongated spheroid. Depending on the variety, the weight of the fruit may vary from 3 to 10 g. During ripening, the colour changes from green to purple or black. The pulp is very bitter, especially during maturation, and comprises between 70 to 90% of the fruit, the bitter taste is due to the presence of a substantial quantity of phenolic compounds (Amiot et al. 1986; Vlahov 1992). The mature seed is very tough and elongated. The olive fruit is rich in oil which originates from the pulp; most cultivated olives have an oil content of 22% by fresh weight (Kiritsakis and Markakis 1987). The fruit is the raw material for a variety of products including olive oil and table olives. The preparation of table olives is a complex industrial process which involves fermentation and/or alkali treatment (Fernandez-Diez et al. 1985). The pulp of the olive fruit is composed of different tissue types (Fig. 1) and there is a paucity of information on the cell walls of these tissues. A knowledge of the structure and chemical composition of olive cell walls is crucial for a better understanding of the biochemical changes that occur in olives during growth, maturation and processing. This chapter describes methods that can be used for the isolation of relatively pure cell walls from olive pulp and the extraction of cell wall polysaccharides by methods that cause minimum degradation. For detailed structural studies, cell wall material virtually free of intracellular compounds is required. However, for certain practical purposes, an alcohol-insoluble residue (AIR) may suffice, provided the limitations of the preparation are borne in mind. The methods used for the separation and characterization of the polymers are described briefly, with particular reference to some of the more recent developments. The experimental details described below are mainly based on our work on the isolation analysis of cell walls of olive pulp (Coimbra 1993; Coimbra et al. 1994).

Isolation and characterisation of cell wall polymers from olive pulp (Olea europaea L.)

Abstract Cell wall material (CWM) was prepared from olive pulp, which is rich in oil. Polymers were solubilised from the CWM by sequential extraction with trans -1,2-cyclohexanediamine- N,N,N′,N′ -tetraacetic acid, sodium salt; Na 2 CO 3 ; 1 M KOH; 4 M KOH; and 4 M KOH+borate to leave the cellulose-rich residue (CR). A suspension of this residue, on neutralisation, released a pectic material, to give residue CR1. The residue CR1 contained cross-linked pectic polysaccharides and some cell wall glycoproteins which were solubilised with chlorite-acetic acid. The polymers from the various extracts were fractionated by graded precipitation with ethanol prior to anion-exchange chromatography, and selected fractions were subjected to methylation analysis. Closely related pectic polysaccharides rich in arabinose were the major components of the cell walls and they differed in their ease of extraction from the wall matrix. Significant amounts of acidic xylans were isolated from the precipitates obtained on neutralisation of the 1 M KOH extracts, and a large proportion of these would have been derived from the lignified sclereids; the xyloglucans from the supernatant fractions had xylans associated with them. However, significant amounts of xyloglucans associated with glucomannans, but virtually free of xylans, were isolated from the ‘neutral fractions’ of the supernatant solutions from the 4 M KOH and 4 M KOH+borate extracts. These ‘neutral fractions’ also contained significant amounts of hydroxyproline-rich cell wall glycoproteins. The anomeric configurations of the sugars in the glucuronoxylans were determined by 13 C NMR spectroscopy. The structural features of the wall polymers are discussed in relation to the structure of the cell wall of the fruit pulp.

Morphogenesis Control in Candida albicans and Candida dubliniensis through Signaling Molecules Produced by Planktonic and Biofilm Cells

ABSTRACT Morphogenesis control by chemical signaling molecules is beginning to be highlighted in Candida biology. The present study focuses on morphogenic compounds produced in situ by Candida albicans and Candida dubliniensis during planktonic and biofilm growth that may at least partially substantiate the effect promoted by supernatants in morphogenesis. For both species, planktonic versus biofilm supernatants were analyzed by headspace-solid-phase microextraction and gas chromatography-mass spectrometry. Both planktonic cells and biofilm supernatants of C. albicans and C. dubliniensis contained isoamyl alcohol, 2-phenylethanol, 1-dodecanol, E-nerolidol, and E,E-farnesol. Alcohol secretion profiles were species, culture mode, and growth time specific. The addition of exogenous alcohols to the cultures of both species inhibited the morphological transition from the yeast to the filamentous form by up to 50%. The physiological role of these alcohols was put to evidence by comparing the effects of a 96-h cultured supernatant with synthetic mixtures containing isoamyl alcohol, 2-phenylethanol, E-nerolidol, and E,E-farnesol at concentrations determined herein. All synthetic mixtures elicited a morphological effect similar to that observed for the corresponding supernatants when used to treat C. albicans and C. dubliniensis cultures, except for the effect of the 96-h C. dubliniensis planktonic supernatant culture on C. albicans. Overall, these results reveal a group of alcohol extracellular signaling molecules that are biologically active with C. albicans and C. dubliniensis morphogenesis.

Calcium-mediated gelation of an olive pomace pectic extract

Pectic raw material was extracted from the alcohol insoluble residue of olive pomace. After purification, the olive pectic extract (OPE) contained 48% of galacturonic acid (GalA) and 31% of arabinose, in a total sugar content of 72%, and a degree of methylesterification (DM) of 43%. Phase diagrams were established to define the physical state of the OPE/calcium systems, at pH 3 and 7, as a function of GalA and calcium concentration. The rheological properties of the OPE/calcium systems were investigated by small-amplitude oscillatory tests. Kinetics of gel ageing and the viscoelastic properties of the cured gels were evaluated as a function of GalA and calcium concentration at pH 3 and 7. Compared with the observed behaviour of a citrus pectin, taken as representative of a commercial low-methoxyl pectin, the OPE showed higher critical GalA and calcium concentrations for gelation to occur, a larger region corresponding to homogeneous gel, and gels characterised by lower viscoelastic moduli, at similar GalA and calcium concentrations.

Temperature dependence of the formation and melting of pectin-Ca2+ networks: a rheological study

Abstract Small deformation dynamic rheometry was used to characterise the calcium-induced gelation of low-methoxyl pectins, at two different pH values. The gelation kinetics was interpreted with basis on the change of the storage modulus with time, taken as a measure of changes in cross-linking density within the pectin–calcium network. The temperature influence on the rate of gel formation and ageing was evaluated, as well as the temperature sensitivity of the cured gels. The formation and the softening/melting of pectin–calcium networks were also studied under non-isothermal conditions. The structural diversity of the two pectin samples had a great influence on the gelation kinetics and thermal behaviour of these pectin–calcium networks, due to differences in the steric arrangement or environment and/or availability of the chelating groups. An association mechanism is suggested to predominate under conditions of low availability of dissociated carboxylic groups, due to a low pH, higher degree of methylation, or steric constraints introduced by acetylation or neutral side chains, different from the classical ‘egg-box’ model, and where non-ionic hydrogen bonding and hydrophobic interactions may co-exist and cooperate with coordinative binding of calcium-ions.

Chemical and physical methodologies for the replacement/reduction of sulfur dioxide use during winemaking: review of their potentialities and limitations

Sulfur dioxide (SO2) is probably one of the most versatile and efficient additives used in winemaking due to its antiseptic and antioxidant properties. This compound is also important for minimizing phenolic polymerization rate and color loss during wine aging. However, allergies caused by SO2-derived compounds, namely the sulfites, are becoming more frequent, causing symptoms such as headaches, nausea, gastric irritation, and breathing difficulties in asthma patients. Consequently, the legislated maximum concentration of SO2 allowed in wines has been gradually reduced. For this reason, it is crucial in a competitive global winemaking market strategy, to reduce or even eliminate the use of SO2 as a preservative and to search for new healthier and safe strategies. This work gives an overview of the main methodologies that have been proposed so far and that have potential to be used in winemaking as an alternative to SO2. The addition of compounds such as dimethyl dicarbonate, bacteriocins, phenolic compounds, and lysozyme, and the use of physical methods, namely pulsed electric fields, ultrasound, ultraviolet radiation, and high pressure are discussed and critically evaluated.

Quantification approach for assessment of sparkling wine volatiles from different soils, ripening stages, and varieties by stir bar sorptive extraction with liquid desorption.

Stir bar sorptive extraction with liquid desorption followed by large volume injection coupled to gas chromatography-quadrupole mass spectrometry (SBSE-LD/LVI-GC-qMS) was applied for the quantification of varietal and fermentative volatiles in sparkling wines. The analytical data were performed by using suitable standards of monoterpene hydrocarbons (alpha-pinene), monoterpenols (linalool), sesquiterpenoids (E,E-farnesol, Z-nerolidol, and guaiazulene), C(13) norisoprenoids (beta-ionone), aliphatic and aromatic alcohols (hexanol and 2-phenylethanol), and esters (hexyl acetate and ethyl decanoate) as model compounds. The wine volatiles were quantified using the structurally related standards. The methodology showed good linearity over the concentration range tested, with correlation coefficients ranging from 0.950 to 0.997, and a reproducibility of 9-18%. The SBSE-LD/LVI-GC-qMS methodology allowed, in a single run, the quantification of 71 wine volatiles that can be quantified accurately at levels lower than their respective olfactory thresholds. This methodology was used for assessment of sparkling wine volatiles from different soils, ripening stages, and varieties. The variety and soil influenced significantly the volatile composition of sparkling wines; lower effect was observed for the ripening stage of grapes picked up one week before or after the maturity state.

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans.

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.