Apple Cell Wall Material Research Articles

Role of pectic polysaccharides in structural integrity of apple cell wall material

Texture modulating properties of aqueous dispersions of apple cell wall material differed from those of tomato or kiwifruit, particularly under high shear. It was previously hypothesized that this may be due to the fact that the apple cell wall showed less in vivo solubilization of pectic polysaccharides during ripening compared to tomato or kiwifruit. However, in vitro solubilization of the pectic polysaccharide content of apple CWM by endo-polygalacturonase and/or extraction with 0.05 M sodium carbonate, did not affect the loss in wall integrity shown by tomato or kiwifruit CWMs under shear. In addition, the pectin-depleted residue after Na2CO3 extraction possessed better water retaining and viscosity generating properties than the original cell wall material. Following treatment of apple CWM with cellulase, the viscosity of suspensions decreased, emphasising the role that the cellulose–hemicellulose network plays in the water-retaining capacity of the cell wall. Residue from CWM after cellulase treatment consisted of ∼85% pectic polysaccharides. Surprisingly, the integrity of these “cellulose-free” walls was maintained after shear. It is concluded that differences in structural properties of the CWMs of apple compared to kiwifruit or tomato are not simply related to pectin solubilization but to a fundamental difference in the architecture of the apple cell wall.

Non-covalent interaction between procyanidins and apple cell wall material. Part III: Study on model polysaccharides

The adsorption of condensed tannins (procyanidins) of varying degrees of polymerisation and percentage of galloylation on solid polysaccharides substrates was quantified using the Langmuir isotherms formulation. Pectins and xyloglucans, which are soluble polysaccharides, were first cross-linked by, respectively, dibromopropane and epichlorohydrin to obtain insoluble covalent gels. Cellulose and starch, being insoluble in the buffer solution at room temperature, were used as bought. Apparent affinity constants obtained for the pure polysaccharides were as follows: pectin>>xyloglucan>starch>cellulose. The apparent affinity constants increased with the molecular weight of the procyanidins, except with cellulose. Higher affinities were obtained with pectin, a polysaccharide having the ability to develop a gel-like network, forming hydrophobic pockets able to encapsulate procyanidins. Filamentous and globular polysaccharides, like cellulose and xyloglucan, bound procyanidins weakly. Higher apparent saturation levels were obtained for cellulose and xyloglucans, the arrangement of which would favour cooperativity and stacking. Pectin had lower saturation levels probably due to a steric hindrance effect.

Non-covalent interaction between procyanidins and apple cell wall material. Part II: Quantification and impact of cell wall drying

The adsorption of condensed tannins (procyanidins) on solid cell wall material was quantified using the Langmuir isotherms formulation. Six tannins fractions differing by their size (number average degree of polymerisation between 2.5 and 65) and composition (presence of galloyl groups from to 0 to 22%, proportions of (+)-catechin to (-)-epicatechin from traces to one CAT for three EPI) were used. Two cell walls differing only by their physical characteristics were obtained by mild or harsh drying, with surface areas of 2.15 and 0.52 m(2)/g, respectively. The total amounts of procyanidins retained on the cell wall materials increased with their concentrations while the proportions of retained decreased, and a plateau was reached at high concentrations. The apparent affinity of procyanidins for CWM isolated from apples increased with their molecular weight. Decrease of the CWM porosity by harsh drying slightly decreased the apparent affinity and increased the apparent saturation levels when constants were expressed relative to cell wall weight, but strongly increased both apparent affinity and apparent saturation levels per surface units.

Non-covalent interaction between procyanidins and apple cell wall material: Part I. Effect of some environmental parameters.

The adsorption of procyanidins on cell wall material were quantified by bringing into contact a solution of procyanidins and a suspension of cell wall material. The influence of structural features such as degree of polymerisation (DP) and percentage of galloylation (% gall), and of physico-chemical parameters such as pH, ionic strength, temperature and presence of ethanol were investigated. The amount of procyanidins bound to the cell wall increased with the DP, the % gall, and the proportion of (+)-catechin, the last indicating an effect of the stereochemistry of the flavan-3-ols. Complex formation between procyanidins and cell wall material was not affected by pH in the range 2.2-7 but it was decreased by urea, dioxane and ethanol. Adsorption increased with increasing ionic strength and decreased with increasing temperature. This indicated that the bonds which governed the interaction between procyanidins and cell wall material were weak energy bonds of the type hydrogen bond and hydrophobic interaction.

The influence of suspension solution conditions on the rehydration of apple cell wall material

Dried apple cell wall material, consisting mainly of intact single cells, was rehydrated with water (reference) and diluted solutions of potassium acetate (K), malic acid (A) and fructose (F) as well as aqueous mixtures of these substances. The rehydration process was investigated using the bed volume technique (swelling, including kinetic), filtration methods [water retention capacity, (WRC)], the capillary pressure method (WRC at ambient pressure and WRC at pressure of 1 kPa, including kinetic of water release) as well as by rheological analysis (shear flow and oscillation) of the suspensions formed by rehydration. An experimental design was created with the three factors K, A, F and the obtained data were analyzed by ANOVA (effects of the main factors K, F, A and the interactions KF, KA, FA, KFA). The presence of K in the rehydration solution increased the velocity of swelling, the WRC and the texturization of the formed suspensions, and in contrast, the presence of A decreased these values. There were also found a few interactions of the soluble substances by which the rehydration process was significantly promoted or hindered. The direction of these effects, however, cannot be exactly predicted in each case at present. Therefore, further investigations are necessary to improve the understanding of the multivariate relationships.

Degradation of apple cell wall material by commercial enzyme preparations.

The action of commercial enzyme preparations on the release of cell wall constituents from alcohol-insoluble substance prepared from apples without skins and cores as well as their influence on the water binding of remaining residues is described as a model for the enzymatic cell wall destruction during production of liquid fruit products. Besides 'normal' enzyme concentrations adapted from the usual industrial dosage, 'tenfold' enzyme concentrations were applied. Dependent on enzyme spectrum and activities, concentrations of dietary fibre, e.g., pectin, increased in the soluble fractions using conditions of enzymatic 'mash treatment'. A further release of these cell wall constituents occurred when cellulase containing enzyme preparations were used under conditions of 'pomace treatment', especially with the 'tenfold' enzyme dosage. The partial enzymatic degradation of the cell wall material is connected with a decrease in water binding of the remaining residues during both simulated mash treatment of pomace treatment. Alcohol-insoluble substance from apples is a suitable model for the determination of complex enzymatic actions of enzyme preparations containing pectolytic, hemicellulolytic, and/or cellulolytic activities under standardised conditions.

Material properties of processed fruit and vegetables. II. Water hydration properties of cell wall materials from apples

Apple cell wall material was exposed to different processing steps, commonly used in the fruit and vegetable industry, such as heat and enzymatic treatment (maceration, liquefaction) as well as extraction with diluted acid. The influence of these procedures was investigated with respect to chemical composition and structural features and their effect on the material and functional properties. Besides water binding determinations commonly used, kinetic methods were introduced. In this way it was shown that the treatments influence not only the water retention capacity but also the velocities of swelling and water release under pressure, respectively. Heat treated materials with softened cell walls released water slower than cold water extracted materials. Furthermore, it was found that a high pectin content or a remaining natural grown cell structure are no prerequisites for a good water binding ability. The relationships are rather very complex and multivariate and all factors influence mutually.

Effects of commercial pectolytic and cellulolytic enzyme preparations on the apple cell wall.

The action of three different commercial enzyme combinations on apple cell wall material has been examined in a model system under conditions of mash and pomace treatment by using an alcohol-insoluble substance prepared from apples. A part of the total dietary fiber, for example, galacturonan (pectin), appeared in the soluble fraction after enzymatic mash treatment. The soluble fraction increased intensely during pomace treatment. Furthermore, enzyme actions caused a change in the water-binding capacity of residues as well as changes in the monosaccharide composition and in the molecular weight distribution of saccharides in filtrates (soluble parts). The extent of decomposition of cell wall material and the increase of soluble oligomeric and/or polymeric dietary fiber components are caused by both the composition (pectinases, cellulases, and hemicellulases) and the activities of the enzyme preparations. The model experiments allow an insight into the reactions occurring during enzyme action on the plant cell wall, for example, during apple juice production using pectolytic and cellulolytic enzyme preparations.

Apple pomace liquefaction with pectinases and cellulases:analytical data of the corresponding juices

Apple juices were produced by way of a two-stage process consisting of traditional enzyme treatment of the mash with pectinases for the premium juices and pomace liquefaction with different pectinases and cellulases for the extraction juices. Premium and extraction juices were analysed separately. Calculated to an equal juice strength of 12° Brix, there was an increase of D-galacturonic acid and cellubiose in the extraction juices. Released galacturonic acid from cell wall material was found at levels ranging from 107 mg/l to 1239 mg/l. This was an essential contribution to the total titrable acid of the extraction juices. The sum of phenolic substances determined by high performance liquid chromatography was significantly higher in all the extraction juices than in the corresponding premium juices. Among the phenolics, the dihydrochalcone phloretin 2′-glucoside (92–110 mg/l) showed an increase of 4 to 5 times the concentration in the respective premium juices. Quercetin derivatives were mainly present in the extraction juices; here the values were between 32 mg/l and 38 mg/l. Under the influence of strong pectolytic or cellulolytic enzyme activities, oligo- and polysaccharides are released from the apple cell wall material, resulting in colloid concentrations of up to 15 g/l in the extraction juices. High concentrations of polyphenols and pectic polysaccharides can lead to technological problems. However, pomace liquefaction may also turn out to be suitable for obtaining value-added foods. In respect to nutritional aspects, enzymatic treatment of pomace offers the opportunity of releasing apple polyphenols and polysaccharides contained in the pomace to a greater extent and obtaining them preparatively.

Degradation of apple fruit xyloglucan by endoglucanase

A purified, alkali-extractable apple fruit xyloglucan (APfxg) was treated by endoglucanase (endoIV) from Trichoderma viride. The degradation products were fractionated by size-exclusion chromatography on BioGel P-2; the pentamer to dodecamer fractions were further fractionated by semi-preparative high-performance anion-exchange chromatography. The purified oligosaccharides were characterized by monosaccharide analysis, mass spectrometry and degradation with Driselase. Based on these data, tentative structures were proposed for most products. Apple xyloglucan is composed of a diversity of repeating units. Next to the major oligosaccharides XXXG, an octamer, XXFG and XLFG (Renard, C.M.G.C, Lomax, J.A. & Boon, J.J. (1992). Carbohydr. Res., 232, 303–320), XG, XXG, FG and two new fucose-containing oligosaccharide building blocks (hexasaccharide and dodecasaccharide) were found. Larger xyloglucan fragments were obtained by partial degradation of APfxg by endoIV, and treatment of apple cell wall material by endoI. These fragments were predominantly composed of fucose-containing oligosaccharides. Our results show that both endoIV and endoI are hindered by fucosylated side chains.

Studies on apple protopectin. III: Characterization of the material extracted by pure polysaccharidases from apple cell walls

Plant cell walls contain polymers with complex and badly understood interactions that can be studied by enzymic extraction. Apple cell wall material, partially depectinated by a chelating agent, was treated with pure polysaccharidases (pectin-lyase, polygalacturonase, pectinesterase, endo-β-(1,4)-glucanases, exo-β-(1,4)-glucanase, endo-α-(1,5)-arabinanase, arabinofuranosidase, endo-β-(1,4)-galactanase and endo-β-(1,4)-xylanase) and combinations of these enzymes. Glucanases, arabinanases and galactanase extracted neutral oligomers. Enzyme formulations active on highly methylated pectins freed galacturonate oligomers and high-molecular-weight material. Addition of endo-β-(1,4)-glucanase to these enzyme formulations increased the amounts of high-molecular-weight material in the extracts. One of the endo-β-(1,4)-glucanases had a more marked action. The high-molecular-weight material was rich in neutral sugars, notably arabinose and galactose, and was degraded to a large extent by arabinanases and galactanase. The results were analysed by principal component analysis.

Studies on apple protopectin. II: Apple cell wall degradation by pure polysaccharidases and their combinations

Plant cell walls are complex associations of polymers, the interconnections of which can be studied by sequential enzyme degradation. Partially depectinated apple cell wall material was degraded by pure polysaccharidases (pectin-lyase, polygalacturonase, pectinesterase, endo-β-(1,4)-glucanases, exo-β-(1,4)-glucanase, endo-α-(1,5)-arabinanase, arabinofuranosidase, endo-β-(1,4)-galactanase and endo-β-(1,4)-xylanase) and combinations of these enzymes. Amounts of extracted material and composition of the residues were determined. Principal component analysis permitted an objective assessment of these results. Extraction of significant amounts of uronides was only possible with enzyme formulations active on highly methylated pectins. Addition of endo-β-(1,4)-glucanase enhanced cell wall degradation by enzyme formulations active on highly esterified pectins, but arabinanases and galactanase did not increase the extraction of uronides. Principal component analysis indicated that the effects of the endo-β-(1,4)-glucanases were related to degradation of a fucogalactoxyloglucan, and emphasized the specificity of the action of one of the endo-β-(1,4)-glucanases.

Studies on the α-L-Arabinofuranosidase Complex from Sclerotinia fructigena in Relation to Brown Rot of Apple

α-L-Arabinofuranosidase (AF) was detected in apple fruitlets experimentally infected by Sclerotinia fructigena. In extracts of such fruitlets, three AF isoenzymes were separated by preparative isoelectric focusing. When the fungus was grown in shake culture with different carbon sources, AF was detected in each culture filtrate and mycelial homogenate. Although fungus growth and total AF varied with the carbon source, the AF isoenzyme pattern was similar in each instance to that obtained when grown on sodium polypectate. Each of the partially purified AF isoenzymes behaved differently in substrate specificity and inhibitor studies; however, each showed a specificity for α-L-arabino-furanosides. The two extracellular AF isoenzymes released monomeric arabinose when incubated with araban or apple cell walls. External AF III (pI 6·5) was more active on a substrate of apple cell wall material than external AF I (pI 3·0). The latter form of the enzyme was less susceptible to inhibition by either oxidized or unoxidized apple juice. Two isolates of Sclerotinia fructigena with low growth rate in vivo secreted no AF III in vitro.