Extrusion-induced changes to the chemical profile and viscosity generating properties of citrus fiber.

Each of 8 variants in extrusion conditions was applied to a commercially available citrus fiber. Extrusion under conditions where the specific mechanical energy (SME) exceeded 400 kJ·kg(-1) was able to solubilize up to 30% of the fibers. Where the SME was ∼200 kJ·kg(-1) the degree of fiber solubilization was between 8 and 12%. All extruded fibers showed a loss of water-retaining capacity compared to the reference fiber, and this was attributed to the disruption of the integrated cell wall structure during the extrusion process. Nevertheless, within the 8 extruded variants there was a wide range of viscosity generating capacity which depended on the level of SME to which the fibers were subjected. The SME also had a pronounced effect on the nature of the solubilized fibers in terms of both their monosaccharide composition and their molecular weight profile. Both pectic and hemicellulosic polysaccharides were solubilized. It is concluded that extrusion has promise as a physical process for manipulating both the technological functionality and the health promoting properties of dietary fibers.

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans.

The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (1-->5)-alpha-arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.

Purification and characterisation of a galactoglucomannan from kiwifruit (Actinidia deliciosa).

A galactoglucomannan (GGM) has been purified from the primary cell walls of ripe kiwifruit. A combination of barium hydroxide precipitation, anion exchange- and gel-permeation chromatography gave a chemically homogeneous polymer with a 1:2:2 galactose-glucose-mannose ratio and a molecular weight range of 16-42 kDa. Complete hydrolysis of the polymer with endo-1,4-beta-mannanase (EC 3.2.1.78) from Aspergillus niger gave a mixture of oligosaccharides, three of which (II, III, IV) accounted for more than 80% of the GGM. Structural characterisation of these oligosaccharides and the original polysaccharide was achieved by linkage analysis, 1D and 2D NMR spectrometry and enzymatic hydrolysis. Oligosaccharide II beta-D-Glcp-(1-->4)-beta-D-Manp-(1-->, III beta-D-Glcp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Manp-(1-->, and IV beta-D-Glcp-(1-->4)-[beta-D-Galp-(1-->2)-alpha-D-Galp-(1-->6)]-beta-D-Manp-(1-->4)-beta-D-Glcp-(1-->4)-beta-D-Manp-(1-->, appeared in the molar ratio of 2:1:1. A trace amount of mannobiose (I) was detected, indicating that some of the mannosyl residues were contiguous. It is concluded that the predominant structural feature of kiwifruit GGM is a backbone of alternating beta-(1-->4)-linked D-glucopyranosyl and D-mannopyranosyl residues, with approximately one third of the latter carrying side-chains at 0-6 of single alpha-D-Galp-(1--> residues (50% of the branches) or the disaccharide beta-D-Galp-(1-->2)-alpha-D-Galp-(1--> (50% of the branches), the substituted residues being separated by three or five unsubstituted monosaccharide units.

Polysaccharides of green Arabica and Robusta coffee beans.

Two independent procedures for the quantitative determination of the polysaccharide content of Arabica Caturra (Coffea arabica var. Caturra) and Robusta ROM (Coffea canephora var. ROM) green coffee beans showed that they both contained identical amounts of polysaccharide. Cell wall material (CWM) was prepared from the beans and partial solubilisation of component polysaccharides was effected by sequential extraction with water, 1 M KOH, 0.3% NaClO2, 4 M KOH and 8 M KOH. The monosaccharide compositions of the CWMs were similar, although Arabica beans contained slightly more mannose than Robusta. In the latter, more arabinogalactan was solubilised during preparation of the CWM and the water-soluble fraction of the CWM contained higher amounts of galactomannan than in Arabica. Linkage analysis indicated that the galactomannans possessed unbranched to branched mannose ratios between 14:1 and 30:1 which is higher than previously reported. No major difference in the structural features of the galactomannans between species was found. The arabinogalactans were heterogeneous both with regard to the degree of branching and the degree of polymerisation of their arabinan side-chains. Compared to Arabica, Robusta appeared to contain greater amounts of arabinogalactans with longer side chains. It is concluded that there was no detectable difference between the Arabica and Robusta varieties of this study in their absolute polysaccharide content or in the gross structural features of their galactomannans. Differences were apparent both in the structural features and ease of solubility of the arabinogalactans but a more detailed study of several varieties of Arabica and Robusta will be required to determine whether these differences occur consistently between species.

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans.

Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), 0.05 M Na2CO3, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na2CO3, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides.

Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit.

Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA. The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.

Xyloglucan Endotransglycosylase Activity Increases during Kiwifruit (Actinidia deliciosa) Ripening (Implications for Fruit Softening).

The activity of xyloglucan endotransglycosylase (XET) was as-sayed in three tissue zones of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var deliciosa cv Hayward) at harvest and at several softening stages following a postharvest ethylene treatment. At harvest, extractable XET activity per unit fresh weight in the inner pericarp (IP) and core tissue was 4.5 and 42 times higher, respectively, than in the outer pericarp (OP). Within 24 h of ethylene treatment there was an increase in the activity and specific activity of XET in all tissues that continued throughout softening. Activity increased most in the OP, where it showed a 12-fold rise 6 d after ethylene treatment compared with 4.5- and 2.5-fold increases in the IP and core tissues, respectively. Visible swelling of the cell wall in each tissue was observed 24 h after the first detectable rise in XET activity and was most pronounced in the OP, which showed the greatest percentage increase in XET activity. Xyloglucan, galactoglucomannan, and cell wall materials isolated and purified from kiwifruit OP were tested as donor substrates for kiwifruit XET. The enzyme showed activity against xyloglucan but was inactive against galactoglucomannan. XET was active against cell wall materials from unripe and ripe fruit, with swollen walls from the latter being the better substrate. The results indicate that XET may have a key role early in fruit ripening, loosening the cell wall in preparation for further modification by other cell wall-associated enzymes.

Mannitol Synthesis in Higher Plants : Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase.

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase (14)C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K(m) (M6P) was 15.8 millimolar, but the apparent K(m) (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg(2+), Ca(2+), Zn(2+), and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.

Cell Wall Dissolution in Ripening Kiwifruit (Actinidia deliciosa) : Solubilization of the Pectic Polymers.

Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa ;Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na(2)CO(3), 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na(2)CO(3)-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na(2)CO(3)-soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA-soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation. Pectolytic enzymes such as endopolygalacturonase and beta-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo.

Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia.

The rodlet layer of Neurospora crassa macroconidia has been purified and chemically characterized. Sheets of rodlets were released from the conidial surface by vigorously shaking conidia in water. Conidia were removed by filtration and low-speed centrifugation, and the rodlets were recovered from the supernatant by high-speed centrifugation. The rodlet pellet comprised 1.9% of the initial dry weight. Chemical analysis was hampered by the insolubility of the rodlets. They were not solubilized by heating in various protein-denaturing buffers and were only partially dissolved by heating in 1 M NaOH at 100 degrees C for 5 min. Nevertheless, they were found to be largely composed of protein (91%, based on total nitrogen). The major amino acids in acid hydrolysates were aspartic acid, glycine, serine, alanine, half-cystine, and valine. Glucosamine was not detected in acid hydrolysates. The sulfur content was 2.5%, and this could be accounted for in half-cystine and methionine. Carbohydrate comprised just over 2%. The phosphorus content was 0.21%, of which less than one-third was accounted for in phospholipid. The total fatty acid content was 1.0%, most of which could be accounted for by the fatty acids of the phospholipids.