Alain Baron

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.

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.

Composition and properties of purified phenolics preparations obtained from an extract of industrial blackcurrant (Ribes nigrum L.) pomace

Summary An extract obtained from industrial blackcurrant pomace was purified and fractionated on an RP C18 column, resulting in three phenolics preparations of different composition. The phenolics in the preparations were quantified by HPLC, their anti-oxidant capacity was determined, and the preparations were subjected to HPLC-MS analysis in order to identify the bioactive compounds present. The purified preparations selected for study were an anthocyanin-rich preparation, a myricetin and quercetin glycosides-containing preparation, and an aglycon-containing preparation. All three preparations were characterised by having a high content of polyphenols. The anthocyanin-rich preparation was the most concentrated in polyphenols [> 50% (w/w)], followed by the aglycon-containing preparation [> 20% (w/w)], and finally the glycosides-containing preparation [> 9% (w/w)]. The preparations were also characterised by their high anti-oxidant capacity, which was > 3,100 µM TEAC g−1 (TEAC = Trolox Equivalent Anti-oxidant Capacity) for all preparations. The HPLC-MS study confirmed that the anthocyanin-rich preparation was composed of the following anthocyanins: delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside. The aglycon preparation contained four aglycons, of which myricetin and quercetin were predominant, and kaempferol and isorhamnetin were present in lower amounts. The glycosides-containing preparation was found to be the most interesting, since it contained myricetin, quercetin, kaempferol, and isorhamnetin glycosides. Myricetin and quercetin galactosides were also detected in blackcurrant extract for the first time. Moreover, the presence of nitrile-containing compounds, two acylated anthocyanins, and one auron-type compound was detected.

Reduction of colonic inflammation in HLA-B27 transgenic rats by feeding Marie Ménard apples, rich in polyphenols.

Inflammatory bowel diseases (IBD) are immunomediated ailments affecting millions of individuals. Although diet is regarded as an important factor influencing IBD, there are no accepted dietary recommendations presently available. We administered 7.6 % lyophilised apples obtained from two cultivars (Golden Delicious and Marie Ménard, low and high in polyphenols, respectively) to HLA-B27 transgenic rats which develop spontaneous IBD. After 3 months feeding, rats fed Marie Ménard apples had reduced myeloperoxidase activity (3.6 (sem 0.3) v. 2.2 (sem 0.2) U/g tissue; P < 0.05) and reduced cyclo-oxygenase-2 (P < 0.05) and inducible NO synthase gene expression (P < 0.01) in the colon mucosa and significantly less diarrhoea (P < 0.05), compared with control rats. Cell proliferation in the colon mucosa was reduced significantly by feeding Golden Delicious apples, with a borderline effect of Marie Ménard apples. Gene expression profiling of the colon mucosa, analysed using the Whole Rat Genome 4 x 44 K Agilent Arrays, revealed a down-regulation of the pathways of PG synthesis, mitogen-activated protein kinase (MAPK) signalling and TNFalpha-NF-kappaB in Marie Ménard-fed rats. In the stools of the animals of this group we also measured a significant reduction of bacteria of the Bacteriodes fragilis group. In conclusion, the administration of Marie Ménard apples, rich in polyphenols and used at present only in the manufacturing of cider, ameliorates colon inflammation in transgenic rats developing spontaneous intestinal inflammation, suggesting the possible use of these and other apple varieties to control inflammation in IBD patients.

Chemical composition of French and Polish cloudy apple juices.

Summary Cloudy juices from six apple cultivars from Poland (‘Ariwa’, ‘Gold Milenium’, ‘Florina’, ‘Melfree’, ‘Novamac’, and ‘Rajka’) and four French cultivars (‘Ariane’, ‘Chanteline’, ‘Judeline’, and ‘Judor’) were produced and chemically characterised. The analyses encompassed 23 chemical parameters and phenolics profiles. The most important parameter, differentiating cloudy juice from clear juice, was turbidity. Cloudy juices were characterised by having an average total turbidity of 1,210 Nephelometric Turbidity Units (NTU) and a stability of turbidity of 42%. Some of the results deviated from the accepted ranges given in reference values for apple juices in the Code of Practice of the European Fruit Juice Association. This occurred in the cases of simple sugars and saccharose contents of the Polish apple juices (e.g., up to 56.9 mg l–1 of saccharose vs. a maximum value of 30 mg l−1), and for some mineral compounds in the French apple juices (e.g., sodium values up to 10 mg l−1). Large variations were found in case of important healthconferring components. Water-soluble pectin contents varied from 205 mg l−1 for ‘Chanteline’, to 1,289 mg l−1 for ‘Gold Milenium’; while, in the case of phenolic compounds, the range was from 85.7 mg l−1 for ‘Novamac’, to 524.8 mg l−1 for ‘Melfree’.

Tannin-assisted aggregation of natively unfolded proteins

Tannin-protein interactions are essentially physical: hydrophobic and hydrogen-bond–mediated. We explored the tannin-assisted protein aggregation on the case of β-casein, which is a natively unfolded protein known for its ability to form micellar aggregates. We used several tannins with specified length. Our SAXS results show that small tannins increase the number of proteins per micelle, but keeping their size constant. It leads to a tannin-assisted compactization of micelles. Larger tannins, with linear dimensions greater than the crown width of micelles, lead to the aggregation of micelles by a bridging effect. Experimental results can be understood within a model where tannins are treated as effective enhancers of hydrophobic attraction between specific sites in proteins.

Detailed Polyphenol and Tannin Composition and Its Variability in Tunisian Dates (Phoenix dactylifera L.) at Different Maturity Stages

The polyphenol profile of two Tunisian varieties of dates including flavanols, flavonols, flavones, and hydroxycinnamates was characterized. Three tissue zones (flesh, peel, and stone) and three maturity stages were considered. Phenolic compounds were analyzed using reversed phase high-performance liquid chromatography coupled to UV-visible and electrospray mass spectrometry. Procyanidin oligomers and polymers were characterized and quantified using phloroglucinolysis prior to HPLC analysis. Procyanidin polymers based on (-)-epicatechin structure were by far the most concentrated polyphenols in ripe dates, accounting for 95% of total polyphenols with an average concentration of 14 g/kg in the fresh edible parts of the fruit. Interestingly, procyanidins were also highly concentrated in the stones. The concentration and average degree of polymerization (DPn) of the procyanidins decreased according to maturity. Other phenolics, including caffeoylshikimic acid hexoside, caffeoyl-sinapoyl monohexoside and dihexoside, and acetylated flavonols, were tentatively identified for the first time in the fruit.

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.