Catherine M.G.C. Renard

Interactions between apple cell walls and native apple polyphenols: quantification and some consequences

Cell walls were prepared from apple parenchyma by a phenol:buffer procedure. Polyphenols were extracted from freeze-dried apple parenchyma by methanol and water:acetone, and purified by preparative HPLC. Interactions were quantified by bringing into contact suspended cell walls and polyphenol solutions. Hydroxycinnamic acids and (-)-epicatechin did not bind to cell walls. Binding of procyanidins was fast and reached up to 0.6 g per g cell walls. The amounts of procyanidins bound increased with the initial concentration and with DPn. Procyanidins could be partially desorbed by buffer, more being desorbed in the presence of dissolved cell wall polysaccharides. They were totally desorbed using 8 M urea or acetone:water. Higher polymers were bound selectively from procyanidin mixtures, and very high average DPn were obtained in extensively washed complexes. Binding of procyanidins inhibited enzymic degradation of the cell walls.

Interactions between Polyphenols and Macromolecules: Quantification Methods and Mechanisms

Non-covalent and covalent associations of polyphenols with food macromolecules are two of the most fundamental factors affecting the quality of polyphenol-rich food products. This review therefore describes the biochemical bases of associations between polyphenols and macromolecules, that is, proteins and polysaccharides. Our intent is to provide a level of understanding that can be used to underpin future research directions. This will help to resolve existing issues that limit organoleptic and nutritional qualities of polyphenol-rich foods and drinks. It will also allow a better understanding of the functional consequences of these interactions on food/biological systems. The methods used to study non-covalent and covalent interactions are described, and the limiting factors of each method are emphasized. The biochemical mechanisms of interaction between polyphenols and macromolecules are also described. In processed food, non-covalent polyphenol/macromolecule interactions are largely due to weak associations, and result from a combination of hydrogen bonds and hydrophobic interactions. The biochemical mechanisms for covalent interactions involve oxidation of phenolic compounds, whether enzymatically mediated or not, with the formation of o-quinones or o-semi-quinones, or the cleavage of procyanidin interflavanic bonds in acid medium with the formation of carbocations. The effects of factors such as polyphenol structure, macromolecule structure, relative concentrations of both polyphenol and macromolecule, solvent composition, ionic strength, temperature, and pH are discussed.

Studies of the length of homogalacturonic regions in pectins by acid hydrolysis

The different susceptibilities to acid hydrolysis of the glycosidic linkages in a pectin backbone were used to isolate fractions corresponding to the “smooth”, homo-d-galacturonic regions. Pectins from apple, beet, and citrus were de-esterified, and the resulting pectic acids were hydrolysed in 0.1 M HCl at 80°C for up to 72 h. The intrinsic viscosities of the hydrolysates decreased, and two stages could be distinguished. Up to 10 h, there was a fast decrease, corresponding to the cleavage of the more susceptible linkages between l-rhamnose and galacturonic acid residues, followed by a slower stage, corresponding to cleavage of the linkages between galacturonic acid residues. During the course of the reaction, some galacturonic acid and most of the neutral sugars were solubilised, giving two fractions on Sepharose CL-6B. A minor fraction, composed mostly of galacturonic acid and rhamnose, with rhamnose-galacturonic acid ratios of 1:1.5, 1:2.9, and 1:2.1 for apple, beet, and citrus, respectively, eluted at Kav 0.8, and a major fraction, composed essentially of l-arabinose and d-galactose, eluted at the total volume. The acid-insoluble materials represented 84, 66, and 90% of the original pectic acids for apple, beet, and citrus, respectively. They were progressively freed of neutral sugars; after hydrolysis for 72 h, almost pure polygalacturonates (more than 98 mol% galacturonic acid), representing the homogalacturonic regions, were obtained. The molecular weights of these 72-h acid-insoluble materials from apple, beet, and citrus were similar (respectively, 21000, 19000, and 24000), corresponding to lengths of the homgalacturonic regions estimated to be a minimum of 72–100 galacturonic acid residues.

Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5.

The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10-11.

Characterisation and selectivity of divalent metal ions binding by citrus and sugar-beet pectins

Abstract A scale of selectivity for the binding of calcium and some heavy metal ions by citrus and sugar-beet pectins was set up by pH-measurements. The same order of selectivity was found for the two pectins, decreasing as follows: Cu 2+ ∼ Pb 2+ ⪢ Zn 2+ > Cd 2+ ∼ Ni 2+ ≥ Ca 2+ . Binding isotherms for Ca 2+ , Cu 2+ , Ni 2+ , Pb 2+ and Zn 2+ ions have shown a greater binding level when the ionic strength decreased and when the pectin concentration increased in the presence of 0.1 M NaNO 3 . By comparing binding isotherms, the same order of selectivity was found as by pH-studies. Scatchard plots and Hill index evaluation showed for all ions and all pectins anticooperative interactions in water. In the presence of 0.1 M NaNO 3 , citrus pectins displayed cooperative interactions for all metal ions. In contrast, for sugar-beet pectins, cooperative interactions only occured with Cu 2+ and Pb 2+ . With Ca 2+ , Ni 2+ and Zn 2+ sugar-beet pectins displayed Scatchard plots which could not be distinguished from an anticooperative binding. This difference of behaviour could be related to the presence of acetyl groups decreasing the affinity of Me 2+ for sugar-beet pectins.

Structure of the repeating units in the rhamnogalacturonic backbone of apple, beet and citrus pectins

Abstract After controlled acid hydrolysis of de-esterified apple, beet and citrus pectins, a fraction rich in galacturonic acid and rhamnose was isolated and further fractionated by ion-exchange chromatography. The main peaks, representing for each pectin > 30% of the rhamnose-rich fraction, and 50–78% of its rhamnose, were composed of rhamnose and galacturonic acid in equimolar proportions. Gel-permeation chromatography showed series of homologous oligomers with polymerisation degrees of 6 to 20. NMR data indicated a repetitive structure based on the → 4)-α- d -Gal p A -(1 → 2)-α- l -Rha p-(1 → unit, where the reducing ends were rhamnose units and the non-reducing ends galacturonic acid residues. The same rhamnogalacturonan oligomers were obtained from apple, beet and citrus pectins.

Binding of divalent metal cations by sugar-beet pulp

The binding of divalent metal cations (Ca2+, Cd2+, Cu2+, Ni2+, Pb2+ and Zn2+) by sugar-beet pulp was studied. In the presence of 0.1 m NaNO3 the level of metal cation uptake was found to reach its maximum value very rapidly with the speed increasing both with the sugar-beet pulp concentration and with increasing initial pH of the suspension. Using a pH-metric method and by comparing binding isotherms drawn by measuring the free cation concentration after equilibration, a clear scale of decreasing selectivity was found as follows: Cu2+~Pb2+⪢Cd2+~Zn2+ > Ni2+ > Ca2+. Binding followed the Langmuir-type isotherm except for Ca2+. Adsorption may contribute to the binding phenomenon in addition to ion exchange, which may include electrostatic interactions and even chelation in the case of the more strongly bound cations. Sugar-beet pulp, which is cheap and highly selective, therefore seems to be a promising substrate to entrap heavy metals in aqueous solutions.

Structure and properties of apple and sugar-beet pectins extracted by chelating agents

Abstract Pectins were extracted from apple or sugar-beet cell walls (alcohol-insoluble solids) at pH 4.5 and 20°C and at pH 6.5 and 80°C in acetate and phosphate buffer solutions, respectively, and in the same buffers containing cyclohexanediaminetetraacetic acid or ethylenediaminetetraacetic acid. The yields were very different between the two pH and temperature conditions, but variations with the nature of the extractant were small at pH 4.5 and 20°C, and no differences were observed at pH 6.5 and 80°C. The pectins extracted at pH 6.5 and 80°C were richer in neutral sugars and acetic acid. Degradation of the pectins extracted at high pH and temperature was shown by the decrease of the intrinsic viscosity and by the shift of the uronic acid peak toward the total volume in gel-filtration chromatography. These pectins contained a high molecular weight population rich in neutral sugars and a low molecular weight acid population, in contrast to the pectins extracted at low temperature and pH, for which neutral and acidic sugars had similar elution profiles. At pH 6.5 and 80°C, rather extensive degradation of the pectins occurred. Calcium-binding experiments (potentiometry with specific electrode and conductimetry) indicated a statistical distribution of free carboxyl groups. These results do not support the hypothesis that chelating-agent extractable pectins are held in the cell wall by calcium cross-links involving the formation of “egg-boxes”.

Studies on apple protopectin: I. Extraction of insoluble pectin by chemical means

Abstract The pectic material from Golden Delicious apples, strongly bound in the cell walls of parenchymatous tissues (not extractible with Cyclohexane-Diamino-Tetracetic Acid (CyDTA)) was extracted by: (i) successively, hot buffer, hot acid and cold sodium hydroxide, (ii) sodium carbonate (4°C then 20°C) and (iii) chlorite. The pectin extracts were analysed for uronide, neutral sugars and protein contents, and degrees of methylation and acetylation. They were also subjected to gel-filtration and ion-exchange chromatography. Subfractions obtained by ion-exchange were also analysed. Approximately 55% of the pectic material was not extracted by the CyDTA. Hot buffer, hot acid and chlorite extracted small amounts of relatively highly methoxylated and acetylated pectins. The most efficient extractants were the cold carbonate and the cold sodium hydroxide. They extracted saponified pectins that exhibited similar behaviour on gel-filtration and ion-exchange chromatography, showing two subfractions, of which one was rich in neutral sugars and had a high molecular weight. There was no evidence of phenolic interlinkages.

Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix to short-chain fatty acids by human faecal microbiota in vitro

Proanthocyanidins (PAs) in apples are condensed tannins comprised mostly of (−)-epicatechin units with some terminal (+)-catechins. PAs, especially those having a long chain-length, are absorbed in the upper intestine only to a small extent and are passed to the colon. In the colon they are subjected to microbial metabolism by colonic microbiota. In the present article, the ability of human microbiota to ferment apple PAs is studied. Freeze-dried fruit preparations (apple, enzymatically digested apple, isolated cell-walls, isolated PAs or ciders) from two varieties, Marie Ménard and Avrolles, containing PAs of different chain lengths, were compared. Fermentation studies were performed in an in vitro colon model using human faecal microbiota as an inoculum. The maximal extent of conversion to known microbial metabolites, was observed at late time point for Marie Ménard cider, having short PAs. In this case, the initial dose also contributed to the extent of conversion. Long-chain PAs were able to inhibit the in vitro microbial metabolism of PAs shown as low maxima at early time points. Presence of isolated PAs also suppressed SCFA formation from carbohydrates as compared with that from apple cell wall or faecal suspension without substrates. The low maximal extents at early time points suggest that there is a competition between the inhibitory effect of the PAs on microbial activity, and the ability to convert PAs by the microbiota.