Chelator-soluble Pectin Research Articles

Cell wall-degrading enzymes and changes in cell wall polysaccharides during ripening and storage of bush butter (Dacryodes edulis (G. Don) H.J. Lam) fruit

SummaryActivities of cell wall-degrading enzymes and amounts of pectins and hemicelluloses were measured in bush butter fruits at seven stages of ripeness, from unripe to soft fruits. Enzyme assays showed that only polygalacturonase (PG) and pectinmethylesterase (PME) were present in high amounts. The PME activity remained almost constant throughout ripening while that of PG drastically increased. The other enzymes assayed, including cellulases and hemicellulases, appeared to be absent or present only in very low quantities. Chelator-soluble pectin was the only class of polysaccharides that underwent degradation during ripening. The apparent molecular weight of chelator-soluble pectin decreased during ripening while those of alkali-soluble hemicelluloses remained unchanged.

Vase solutions containing sucrose result in changes to cell walls of sandersonia (Sandersonia aurantiaca) flowers

Abstract It is well established that vase solutions containing sugar can improve the vase-life of many cut flower crops. Since cut sandersonia flowers supplied with 2% sucrose are firmer during wilting compared to water-fed controls, we have examined whether the effects of sucrose treatment extend to alterations in cell wall structure in the floral tissues, which may influence the wilting-related flower softening. Mature but not fully opened individual flowers were removed from the stems of sandersonia plants and were fed continuously with either 2% sucrose solution or water for up to 10 days. Sucrose supplementation resulted in decreased amounts of chelator-soluble pectin and increased amounts of Na2CO3-soluble pectin per individual flower, and also changed the molecular size profiles of both these pectin fractions compared to the water-fed controls. The molecular size differences were obvious after 3 days in vase solutions, and diminished with subsequent vase time. Senescence-related galactose loss was delayed in sucrose-fed flowers but there was no difference in the levels of β-galactosidase activity present in these flowers compared to controls. The observed differences in cell wall pectins due to sucrose feeding were not reflected in differences to the overall firmness of pre-senescent flowers (up to day 3). High levels of galactose persisted into the wilting phase when sucrose-fed flowers were firmer than water-fed controls. We conclude that while sucrose induced significant quantitative and qualitative differences in pectin fractions and galactose content, firmness of floral tissue, particularly during senescence, was not governed by these events alone.

Pectic substances from red beet ( Beta vulgaris conditiva). Part I. Structural analysis of rhamnogalacturonan I using enzymic degradation and methylation analysis

Cell wall material from ripe red beet ( Beta vulgaris L. var. conditiva) was isolated as alcohol insoluble residue (AIR). The chelator-soluble pectin obtained by cyclohexane- trans-1,2-diaminotetraacetate (CDTA) extraction of the AIR was fractionated by anion exchange chromatography (AEC). The main fraction was further fractionated by gel filtration chromatography (GFC). Fractions from both chromatographic systems were stepwise degraded by endo-polygalacturonase, endo-β-(1→4)- d-galactanase, endo-α-(1→5)- l-arabinanase and α- l-arabinofuranosidase. Degradation products were fractionated by GFC or by AEC. Polymeric fractions were investigated by methylation analysis after carbodiimide-activated reduction with NaBD 4. Selected fractions were additionally methylated with trideuteromethyliodide to enable the detection of O-methyl substituted sugars. The results indicate that the CDTA-soluble pectins of red beet cell walls are composed of three different sub-units: a homogalacturonan, which accounts for about 75%, a highly ramified rhamnogalacturonan I (RG-I) and a typical rhamnogalacturonan II (RG-II). RG-I consists of a highly ramified backbone composed of nearly equal amounts of rhamnose and galacturonic acid. Side chains, mainly arabinans, galactans and type-II arabinogalactans are attached to the RG-I backbone. Some arabinans are connected via short galactan chains directly or indirectly to this backbone. Type-II arabinogalactans are formed by “inner” chains consisting of (1→3)-linked galactans and short “outer” chains composed of an average number of one to three (1→6)-linked galactose residues. Terminal arabinofuranoses are linked via the O-3-position to galactose residues. Nearly all non-reducing ends consist of glucuronic acid. Approximately 65% of the glucuronic acid residues are substituted by a methyl ether group and approximately 10%, most probably, by a terminally linked rhamnose.

A Comparison of Two Methods and the Effect of Cooking Time on the Extractability of Pectin from the Cell Walls of Cooking Banana

Two methods to prepare pectin samples from cooking bananas were evaluated by a comparison of pectin molecular weights, levels and solubility. The hot alcohol refluxing method (referred to as hot AIR) caused a reduction in the molecular weight of chelator-soluble pectin in raw banana pulp compared to the method that used cold phenol–acetic acid–water (PAW) to extract the pectin. In addition, the use of hot alcohol resulted in a more than fourfold increase in the amount of water-soluble pectin, and nearly twofold decrease in both chelator- and Na2CO3-soluble pectin in the raw banana pulp tissues compared to the cold PAW method. Using the hot AIR for cell wall fractionation studies to correlate the pectin changes to the thermal softening of banana could lead to misleading conclusions. However, the total content (sum of water-, CDTA- and Na2CO3-soluble pectins) was not altered by these two different preparation methods.

PHYSICOCHEMICAL FACTORS AFFECTING FIRMNESS OF PASTEURIZED JALAPENO PEPPER RINGS

ABSTRACTPhysical and chemical properties that influence textural properties of pasteurized pepper rings of four jalapeno pepper cultivars were determined. Hardness, cohesiveness, gumminess and chewiness were positively correlated with moisture and wall thickness, and negatively associated with water soluble pectin and alcohol insoluble solid (AIS) contents. After 90 days' storage, Veracruz peppers were firmer than the other cultivars due to their greater levels of chelator soluble pectin and bound calcium, and lower levels of water soluble pectin. Texture measurement of fresh peppers was not a reliable predictor of firmness for pasteurized peppers.

Cell wall modifications, degrading enzymes and softening of carambola fruit during ripening

The fruit extracts of the slow-ripening cv. B 10 carambola (Averrhoa carambola L.) contained a number of cell wall hydrolases. The predominant ones appeared to be β-(1,4)-glucanase (as carboxymethylcellulase), pectinesterase, β-galactosidase, and polygalacturonase (PG). Other significant hydrolases the activity of which also increased with ripening were the glycosidases, α-arabinosidase, α-galactosidase, and α-mannosidase, and also the glycanases, β-(1,4)-galactanase and xylanase. Throughout ripening, as pectins and hemicelluloses were being differentially modified, the levels of buffered-phenol cell wall materials, total polyuronides as well as arabinose, galactose, xylose, and glucose decreased. At early ripening phase (days 0–12) there was no apparent pectin solubilization, and the loosely-bound water- and chelator-soluble pectins were the first pectic polysaccharides to be affected. That of the former exhibited an upshift, whilst the latter, showed a distinct downshift in their molecular size profiles. At late ripening phase (days 12–24) when tissue firmness had declined substantially, dramatic changes involving pectins and hemicelluloses were evident. Pectins were solubilized, and this increased solubility was accompanied by depolymerization of all pectin classes and a decrease in the level of the Na2CO3-soluble polyuronides. Coincident with this marked modifications of the tightly-bound, predominant polyuronide fractions and hemicellulose was the increase in activities of polygalacturonase and β-(1,4)-glucanase, suggesting that these enzymes may contribute to wall modifications late during ripening. Some of the other wall hydrolases, namely, α-arabinosidase, α-galactosidase and certain isoforms of β-galactosidase/galactanase, might be relevant to the early ripening changes when pectin solubilization was limited.

Effect of calcium treatment prior toBotrytis cinereainfection on the changes in pectic composition of grape berry

The influence of calcium on the process of infection byBotrytis cinereawas studied following calcium infiltration of grape berries, using Sauvignon, a cultivar ofVitis viniferaknown to be susceptible toB. cinerea.Calcium pretreatment had no significant effect on infection rate. Analysis of pectic fractions of rotten berries showed that total amount of pectins decreased during infection. The proportion of water and chelator soluble pectins increased over the same time period. These changes in pectic fractions were less pronounced when grape berries were calcium infiltrated prior to infection. Demethylated pectins produced by the fungal pathogen are probably protected by a rapid calcium chelation.

Firmness and Cell Wall Characteristics of Pasteurized Jalapeño Pepper Rings as Affected by Preheating and Storage

ABSTRACTChanges in cell wall pectic substances, degree of pectin methylation, bound calcium and firmness were determined in preheated and nonpreheated fresh pack jalapeño pepper rings stored for 5 mo. Pepper rings preheated for 60 min at 50°C in a brine solution (8% NaCl and 0.2% CaCl2, were firmer, had more nonextrctable pectins (NXP), more bound calcium, less water‐soluble and chelator‐soluble pectins (CSP), and less pectin methylation than nonpreheated pepper rings. The greater formation and maintenance of nonextractable pectins, which resisted acid hydrolysis during pasteurization and storage is probably an important factor in firming.

Influence of calcium pretreatment on pectic substance evolution in cucumber fruit (cucumis sativus) duringbotrytis cinerea infection

The role of calcium in protecting plant tissue from infection byBotrytis cinerea was studied in cucumber fruit. A self-defense reaction, consisting of an increase in cell-wall-bound Ca, occurred in the plant tissue after an injury or an infection. However, it was not sufficient to protect the tissue from the spread of the fungus, which is accompanied by pectic substance digestion. The degradation induced mainly a decrease in acid- and chelator-soluble pectin levels, and an increase in water-soluble pectin. Ca treatment of the fruit prior to infection could amplify the cell-wall-bound Ca and therefore lessen pectin digestion by fungal pectinolytic enzymes.

Cell Wall Changes Associated with Normal and Mealy Fruit Softening for a Novel Peach Genotype

The `Stony Hard' gene of peach conferred a unique ability to manipulate softening and textural properties of the fruit by controlling the concentration and duration of exposure to ethylene. Fruit ripened in ethylene-free air softened very slowly. Exposure of fruit to 1 ppm ethylene continuously for 48 h, or discontinuously at 100 ppm over the same time period, significantly accelerated softening—to a normal texture. Exposure of fruit to 100 ppm ethylene continuously for 48 h induced softening to the same level, but to a mealy texture. We have prepared cell walls and conducted sequential chemical extractions from fruit exposed to the ethylene treatments above. Galacturonic acid content of chelator soluble pectin fractions decreased for mealy fruit, compared to fruit with normal texture, indicating that selective pectin degradation was associated with mealiness. Other differences in polysaccharide sugar composition and apparent molecular size associated with slow, accelerated, and abnormal softening in peach fruit will be addressed.

Compositional changes in cell wall polymers during mango fruit ripening

Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.

Impact of Heating on Carrot Firmness: Changes in Cell Wall Components

The impact of heating in boiling water on tissue firmness retention of the carrot genotypes Danvers and B9304 was measured. Both genotypes showed a rapid early loss in firmness that was followed by a slower, more prolonged, softening. Softening was greater in Danvers carrots. Cell wall carbohydrate compositions of the two varieties were similar, but wall component dissolution (solubilization and depolymerization) was more extensive in Danvers. The breakdown of soluble and wall-bound pectins involved β-elimination reactions. A survey of several carrot genotypes indicated that those carrots with chelator-soluble pectins of relatively lower molecular weight tended to soften more extensively during heating

Firmness and Cell Wall Characteristics of Pasteurized Jalapeño Pepper Rings Affected by Calcium Chloride and Acetic Acid

ABSTRACTPepper rings packed in brine containing CaCl2 were firmer, had higher bound calcium, chelator soluble pectin and pectin DE, and less water‐soluble pectin (WSP) than peppers packed in brine containing no CaCl2. Pepper rings packed in low acid brines (1% and 1.2% acetic acid) were firmer and had less WSP than those packed in high acid brine. Those samples (4% acetic acid) resulted in softening and pectin solubilization, but CaCl2 resulted in less softening. Monosaccharide composition of cell walls was not affected by CaCl2 or acetic acid. Firmness retention in CaCl2 treated samples was probably due to greater association between calcium ions and pectic substances, which resisted acid hydrolysis.

926 PB 497 SOFTENING OF GAMMA-IRRADIATED TOMATO FRUITS

The use of irradiation to increase longevity and quality of horticultural commodities often results in undesirable softening. The biochemical basis of irradiation-induced softening is not well understood. In this study, we investigated the role of the pectic polysaccharides in irradiation-induced textural changes of `Sunny' tomato fruit. `Sunny' mature-green and pink fruit subjected to 84 or 240 Krad experienced a dosage-dependent decrease in firmness, an increase in electrolyte leakage, and an increase in chelator-soluble pectins. Additionally, pectins prepared from 240 Krad-irradiated fruit were of markedly lower mol wt compared to those from nonirradiated fruit. Irradiation-induced downshifts in pectin mol wt were also noted for preripe fruit that lack PG activity. Mol wt decreases noted for pectins from 240 Krad-treated fruit exceeded those observed for fully ripe, nonirradiated fruit The role of other cell wall polymers in irradiation-induced textural changes is currently being addressed.

269 CELL WALL CHANGES IN RIPENING NASH1 (HOSUI) FRUIT

Cell wall changes during ripening have a major effect on fruit texture. The cell walls isolated using phenol-Tris buffer were sequentially extracted to give polysaccharide fractions that contained mainly water-soluble pectin, chelator-soluble (CDTA) pectin, hemicelluloses (0.05 M Na2CO3 followed by 1M and 4M KOH) and cellulose. The fractions were analyzed colorimetrically for uronic acid, total neutral sugar and cellulose contents. The component sugars of each fraction were determined as their alditol acetates by GC. Then was a decrease in the two pectin fractions during ripening. The pectins appear to have arabinan and galactan side chains. Pectic galactose decreases during ripening. The weight of the combined hemicellulose fractions did not change during ripening, nor did the cellulose level. At least two types of arabinan are present. Pectins were found in all cell wall fractions. Nashi cell walls contain a relatively large amount of xylan compared to other fruit.

SEASONAL CHANGES ON THE QUALITY OF `PERLETTE' TABLE GRAPES IN RESPONSE TO WATER-DEFICIT STRESS AT PARTICULAR STAGES OF DEVELOPMENT.

The nature of yield and quality losses for many fruits is dependent upon the time when water deficits occur in relation to fruit development. We evaluated the water stress effect applied at different stages of development on the quality and harvest date of 'Perlette' table grapes. Nine treatments based on amount of water applied by the grower (100%, 80% and 60%) and their combinations at two growth stages, fruit set to vérasion (F-V) and vérasion to harvest (V-H) were compared. Weekly samples of fruit were analyzed for diameter, weight, volume, soluble solids, tritatable acidity, pH, and pectin content. Harvest percentage at each date was used as precocity index. Significant differences were found on diameter and weight among treatments at various stages of development. The earliest and highest harvest percentage was for (F-V 80% and V-H 100%) treatment. High °Brix were obtained when the water volume was reduced after vérasion, but quality was poor. Chelator soluble pectins represented > 90% of total soluble pectins and was higher on the treatment (F-V 60% and V-H 60%) at harvest.

Cell Wall Modification during Ripening of `Keitt' and `Tommy Atkins' Mango Fruit

`Keitt' and `Tommy Atkins' mango (Mangifera indica L.) fruit were evaluated for selected ripening criteria at six ripening stages, from mature green to overripe. `Tommy Atkins' mangos developed more red and yellow pigmentation (CIE a* and b*) in peel and mesocarp tissues than `Keitt'. The outer mesocarp of `Keitt' remained firm longer than `Tommy Atkins', and the inner mesocarp was softer than the outer at each stage in both cultivars. Cell wall neutral sugars, particularly arabinosyl, rhamnosyl, and galactosyl residues, decreased with ripening in both cultivars. `Keitt' had more loosely associated, chelator-soluble pectin, accumulated more soluble polyuronides, and retained more total pectin at the ripe stage than `Tommy Atkins'. Both cultivars had similar polygalacturonase (EC 3.2.1.15) activity which increased with ripening. The amount and molecular weight of cell wall hemicellulose decreased with ripening in both cultivars. These data indicate that enzymatic and/or nonenzymatic processes, in addition to polygalacturonase activity, are involved in the extensive softening of mango fruit.

STUDIES ON RHAMNOGALACTURONASE IN FRUIT

Rhamnogalacturonase (RGase) is a new fungal enzyme which degrades the highly branched regions of apple fruit cell wall pectin by cleaving the glycosyl linkage between rhamnosyl and galacturonosyl residues (Schols et al., 1990. Carhohydr. Res. 206:105.). This enzyme, if present in fruit, could play a significant role in fruit softening. Partial purification of RGase was accomplished from a fungal enzyme preparation (Pectinex Ultra SP-L, NOVO Ferment) produced from Aspergillus niger. The crude enzyme hydrolyzed chelator-soluble pectin from red ripe tomato fruit. Methylation linkage analysis of the product suggested that an increase in terminal-rhamnosyl residues accompanied pectin hydrolysis, indicative of RGase activity. Cross-linked alginate, hydroxyapatite, and DEAE-Sephadex chromatography were used to partially purify RGase. Polygalacturonase was efficiently removed using the alginate column. Crude pectin obtained from mature-green tomato fruit cell wall by extracting with 0.5 M imidazole buffer (pH 7) and 50 mM Na-carbonate was incubated with pure polygalacturonase and the residue hydrolyzed with 0.1 N trifluoroacetic acid. This modified pectin was used as a substrate to investigate the presence of RGase in tomato and other fruit.

Changes in Pectic Fractions during Ripening- of Cider Apples

Water-soluble pectin (WSP), chelator-soluble pectin (CSP), and hydrochloric acid-soluble pectin (HASP) were monitored in five Asturian apples (Malus domestics Borkh.) throughout ripening. The alcohol-insoluble solid content was found to decrease during ripening, while those of the WSP and CSP fractions increased in the final stages of ripening. This increase was probably at the expense of the HASP content, which had decreased by the end of the ripening period.

POSTHARVEST CHANGES OF PECTIC SUBSTANCES IN CHILLED PEACHES

ABSTRACT Red Globe peaches were harvested at the threshold-mature stage and either stored at OC after preripening at 20C for 48 h or stored directly at 2C. Total pectin increased from 7.49 mg/g fresh weight in the threshold-mature sample to 10.21 mg/g in pre-ripened fruit stored at OC and 12.83 mg/g in fruits stored directly at 2C. Depending on extraction and storage protocol, between 26% and 73% of total pectin was solubilized. Chelator-soluble fractions had a low degree of esterification (DE ca. 20%), whereas other fractions were highly methylated (DE ca. 56% to 74%). Average DE did not vary between samples. DEAE-cellulose chromatography revealed an uneven distribution of methoxyl groups. Two prominent peaks were observed in chromatograms of chelator-soluble extracts from threshold-mature fruit and fruit stored directly at 2C. One peak was observed in chelator-soluble pectin from preripened fruit stored at OC.