Jean-Francois Drilleau

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.

Characterization of highly polymerized procyanidins in cider apple (Malus sylvestris var. kermerrien) skin and pulp

Abstract Freeze-dried materials of cider apple pulp and skin were submitted to three successive solid-liquid extractions. The water-acetone extracts contained significant amounts of condensed tannins; thioacidolysis revealed that they were a mixture of highly polymerized procyanidins mainly constituted of (−)-epicatechin units. Electrospray ionization mass spectrometry showed a complete series of polymeric procyanidins with a degree of polymerization up to 17.

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.

Purification, properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr-Sepharose®-PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as Km = 0.098 mg ml−1 and Vmax = 3.86 µmol min−1 ml−1 of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry

Characterisation and enzymatic hydrolysis of cell-wall polysaccharides from different tissue zones of apple

Abstract Cell-wall polysaccharides from different tissue zones, namely epidermis, the outer parenchyma, the parenchyma of the carpels zone, the carpels and the core line, were isolated as alcohol-insoluble solids (AIS), extracted with cyclohexane-diaminotetraacetic acid, then hot dilute hydrochloric acid, and analysed, then purified by ion-exchange chromatography. In both zones of parenchyma, the cell-wall material represented about 80% of the total cell-wall material from the whole fruit. The pectins from the outer parenchyma accounted for 70% of the total, whereas the carpels zone contained a large proportion of cellulose and hemicelluloses. However, there was no change in galacturonic acid concentration. The enzymatic solubilisation of tissues or AIS was higher in the parenchyma zones than in the others. Nevertheless, the depolymerisation of the soluble pectins from parenchyma zones with an endopolygalacturonase required the action of pectin methylesterase. The depolymerisation of pectins from the other zones, however, did not.

Effect of oxidative browning of apple pulp on the chemical and enzymatic extraction of cell wall polysaccharides

Abstract Cell wall polysaccharides from cider apple pulp which is non-oxidized or oxidized for 2 min or 4 h were isolated as alcohol-insoluble solid (AIS) or water-insoluble solid (WIS). They were successively extracted with water, ammonium oxalate, hot dilute acid, cold dilute alkali and concentrated alkali or by enzymes such as pectin methylesterase and polygalacturonase together with endoglucanase alone or a combination of pectinases and cellulases. The soluble fraction of pectins decreased with the time of oxidation, especially with the presence of oxidized polyphenols (dark brown pigments) in cell wall materials. The results indicated a hindrance of cell wall polysaccharide hydrolysis by the oxidized polyphenols, but not enough to explain the limited yield using enzymatic treatment of oxidized apple pulp. The different behaviours of apple AIS and WIS towards extraction were discussed.

Effect of storage of apple on the enzymatic hydrolysis of cell wall polysaccharides

Abstract Cell wall materials in the form of water-insoluble solids (WIS) and water-soluble fractions (WSF) were prepared from apples stored at 4 °C for 30 weeks. During storage, the WIS content decreased whereas the WSF content remained unchanged. The total amount of polysaccharides decreased, in particular the pectic polymers which decreased by 10%. In contrast, the soluble pectic fraction increased by 40% whilst its degree of methoxylation remained constant. The arabinose and galactose content progressively declined. The enzymatic treatment of the apple tissues was more effective the longer the storage; yields correlated well with the enzyme hydrolysis of WIS. The accessibility of pectin to poly-galacturonases in apple tissues is discussed since it was higher at the end of storage, whereas the solubilisation of pectins from WIS by polygalacturonases remained constant. On the other hand, with liquefying enzymes, the yield of pectin solubilisation from apple tissues or WIS were well correlated and increased with storage time.

Methods for Determining the Degree of Polymerization of Condensed Tannins: A New 1H-NMR Procedure Applied to Cider Apple Procyanidins

The degree of polymerization (DP) that corresponds to the number of flavan-3-ol units is one of the most important features that characterize condensed tannins (proanthocyanidins) because of its direct link to the various properties of this kind of phenolic compound. In their definition of vegetable tanning substances, Bate-Smith and Swain1 referred to the molecular weight that must range from 500 to 3,000. By considering their extraction, their biological activities, their sensory effects, condensed tannins often behave according to their molecular weight, although this single feature is quite insufficient to show evidence of all their properties. On the whole, the molecular weight of condensed tannins is related to their ability to associate with proteins and polysaccharides; this “tanning capacity” varies in an increasing order with the DP.2–6 This property is also related to other applications. For instance, the work of Lea and Arnold7 pointed out the influence of the DP of procyanidins in relation to bitterness and astringency of cider. Proanthocyanidins are also partly involved in haze formation in beers: the capacity of beer tannins to precipitate proteins increases with the DPn.4,5,8 Many studies dealing with the biological activities of proanthocyanidins also show that antioxidant,9 antifungal,10 anti-enzymic,11 antisecretory,12 or antitumor13 activities may correlate with the DP.

ENZYMATIC MACERATION OF APPLE PARENCHYMA TISSUE. I. MODELLING OF THE KINETICS OF HYDROLYSIS

During the maceration of apple parenchyma with polygalacturonase (PG), three fractions were obtained (residual tissue, free cells or pulp, juice) in which the cell wall material was measured as alcohol-insoluble solids (AIS). A model is proposed to describe the distribution of cell wall material in these three fractions during the course of the enzymatic maceration process, which fitted the experimental data with a determination coefficient r2 superior to 0.99. Two processes of maceration were shown to be involved. A first, fast process, degraded a small part of the tissue while the second process slowly degraded the remaining tissue. The maceration products were principally non degradable cells and non degradable soluble pectins. The degradable cells and the degradable pectins accumulated only at the beginning of the maceration. The rate constants followed the Arrhenius law. The initial rate of maceration increased with the amount of PG or the surface area to reach a limit value at high enzyme concentrations or high surface areas, respectively.

ENZYMATIC MACERATION OF APPLE PARENCHYMA TISSUE. II. EFFECT OF THE DEGREE OF RIPENESS ON THE KINETIC BEHAVIOUR

Apples of four degrees of ripeness were selected to investigate the effect of ripening on maceration of apple tissue by polyglacturonase (PG) using the model proposed to describe the kinetics of maceration [1]. This model permitted to describe the kinetics for all four samples but the precision of the adjustment showed a slight diminution with the degree of ripeness. The proportion of apple tissue macerated by the fast (process 1) and slow (process 2) processes was the only parameter of the model which changed with the degree of ripeness. Analysis of the maceration products (pulp and juice) revealed that these two fractions were gradually degraded during the maceration time.