Environmentally friendly preparation of pectins from agricultural byproducts and their structural/rheological characterization

Apple pomace which is the main waste of fruit juice industry was utilized to extract pectins in an environmentally friendly way, which was then compared with chemically-extracted pectins. The water-based extraction with combined physical and enzymatic treatments produced pectins with 693.2 mg g⁻¹ galacturonic acid and 4.6% yield, which were less than those of chemically-extracted pectins. Chemically-extracted pectins exhibited lower degree of esterification (58%) than the pectin samples obtained by physical/enzymatic treatments (69%), which were also confirmed by FT-IR analysis. When subjected to steady-shear rheological conditions, both pectin solutions were shown to have shear-thinning properties. However, decreased viscosity was observed in the pectins extracted by combined physical/enzymatic methods which could be mainly attributed to the presence of more methyl esters, thus limiting polymer chain interactions. Moreover, the pectins which were extracted by combined physical/enzymatic treatments, showed less elastic properties under high shear rate conditions, compared to the chemically-extracted pectins.     

Pectin Esterification Degree in the Bioavailability of Non-heme Iron in Women

Pectins are a type of soluble fiber present in natural and processed foods. Evidence regarding the effect of esterification degree of pectins on iron absorption in humans is scarce. In the present study, the effect of pectins with different degrees of esterification on non-heme iron absorption in women was evaluated. A controlled experimental study was conducted with block design, involving 13 apparently healthy, adult women. Each subject received 5 mg Fe (FeSO₄) without pectin (control) or accompanied by 5 g citrus pectin, two with a low degree of esterification (27 and 36%), and one with a high degree of esterification (67 to 73%), each on different days. Each day, the 5 mg Fe doses were marked with radioactive ⁵⁹Fe or ⁵⁵Fe. Radioactivity incorporated into erythrocytes was determined in blood samples 14 days after the marked Fe doses were consumed. On days 18 and 36 of study, 30 and 20 mL blood samples were obtained, respectively, and blood sample radioactivity incorporated into erythrocytes was determined. Body iron status was determined from blood taken on day 18. Whole body blood volume was estimated for calculate iron bioavailability; it was assumed that 80% of absorbed radioactivity was incorporated into the Hb. All women participants signed an informed consent of participation at baseline. Iron bioavailability (mean geometric ±1 SD) alone (control) was 18.2% (12.3–27.1%), iron + pectin27 was 17.2% (10.2–29.2%), iron + pectin36 was 15.3% (9.5–24.6%), and iron + pectin67 was 19.5% (10.0–38.0%). No statistically significant differences between iron bioavailability (repeated measures ANOVA, p = 0.22) were observed. Pectin esterification degree does not influence the bioavailability of non-heme iron in women.     

The proportion of calcium-bound pectin in plant cell walls

The amount of pectin held in cell walls by ionic bonds only was determined by extraction with cyclohexanediamine tetraacetic acid (CDTA) at room temperature, to remove calcium ions without degrading the galacturonan chains. Enzymic degradation was avoided by extracting the cell walls with phenol-acetic acid-water during preparation. From cell walls of celery petioles, cress hypocotyls and tomato and cucumber pericarp CDTA extracted 64–100 mg g^-1 pectin, leaving 80–167 mg g^-1 uronic acid in the residue. An additional extraction at high ionic strength was used to make the galacturonan chains more flexible and thus detach any pectins held by steric interactions, but the amount released in this way was small. Most of the residual uronic acid polymers could be extracted by cold alkali and remained soluble on neutralisation, showing that it was not water-insolubility that prevented their extraction with CDTA. Covalent bonding was thought more likely.     

Hemolytically active components from P. parvum and G. breve toxins

Hemolytically active fractions were isolated from the toxins produced by the red-tide dinoflagellate $\text{Gymnodinium breve}$ (GBTX) and the chrysomonad $\text{Prymnesium parvum}$ (PPTX). High pressure liquid chromatography through bonded phase (ODS) silica columns using a gradient of methanol in chloroform yielded 6 major fractions from GBTX, 3 of which were hemolytic (HD₅₀=0.3?0.56 μg·ml^?1). None were ichthyotoxic. Of the 6 fractions obtained from PPTX, 4 were hemolytic (HD₅₀=0.013?2.8 μg·ml^?1) but only one (fraction 6) was ichthyotoxic. This fraction was ～ 2000 times more hemolytic than the crude PPTX (HD₅₀=33.2 μg·ml^?1). Analysis of their UV spectra indicates that the fractions within each group are closely related.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Spore Production Under Solid-State Fermentation of Lignocellulosic Residues

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47?×?10¹⁰ spores g^?1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82?×?10¹⁰ spores g^?1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH₂PO₄, 1 g MgSO₄·7H₂O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105?×?10¹⁰ spores g^?1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10?×?10¹¹ spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.     

Production of extracellular pectinase enzymes by a mutant (Pol6) of Penicillium occitanis

Summary Penicillium occitanis strain Pol6, a mutant developed for hyperproduction of cellulase and pectinase enzymes was used for the study of extracellular pectinase production when pectins from different sources (apple and citrus) and with varying degree of esterification (DE) were used as inducers. Highly esterified citrus pectins were found to be suitable substrates for polygalacturonase, pectinase and pectin methyl esterase production, while low esterified citrus pectin favoured pectin lyase (PL) production. Apple pectins induced other hydrolytic enzymes (e.g., -1,3-glucanase, -glucosidase, -galactosidase), in addition to pectolytic enzymes. Moreover, the combination of high and low esterified citrus pectins induced the production of a complete pectinase complex. The extent of degradation of the substrate and the affinity for PL decreased with decreasing DE irrespective of the source. There was no evidence of PL activity in this strain. No significant effect of cations (Ca⁺⁺, Mn⁺⁺, Na⁺) on PL activity was observed. However, EDTA (100 mm) inhibited 50% of the activity, when tested on highly esterified (rapid set citrus) pectin. Offprint requests to: S. Jain     

Continuous-flow enrichment of a strain ofErwinia carotovora having specificity for highly methylated pectin

A pectinolytic bacterium was isolated from a mixed microbial population by means of a chemostat enrichment procedure. The bacterium, which was identified asErwinia carotovora, grew only on highly methylated pectin and produced a pectin lysase which released unsaturated monomer and dimer from 71% esterified citrus pectin. The pectin lyase was inducible only by pectins having a high methyl content and in pectin-limited chemostats its synthesis passed through a maximum at a dilution rate close to 0.04h^-1.     

Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase

A method was developed that enabled the study of non-esterified galacturonic acid sequences (so-called blocks) in pectin. Endopolygalacturonase of Kluyveromyces fragilis was used to extensively degrade pectin, and the composition of the galacturonic acid molecules produced was determined with high-performance anion-exchange chromatography at pH 5. With this technique, the amount of non-esterified mono-, di-, and trigalacturonic acid released was determined. In addition, the relative amounts of methyl-esterified oligomers--up to 10 galacturonic acid residues could be observed. By comparing the percentages of non-esterified mono-, di-, and trigalacturonic acids released, pectins with large enzyme-degradable blocks could be distinguished from pectins with small enzyme-degradable blocks. High percentages of mono- and digalacturonic acid were found for pectins containing small non-esterified blocks. The total area of all peaks corresponding to methyl-esterified oligomers was found to be indicative for the distribution of these blocks. The higher the ratio of the methyl- to non-esterified peak areas, the more closely associated blocks are present. Randomly esterified pectins, with degrees of methyl esterification of 50 and higher, contained smaller, more clustered blocks than commercial extracted pectins of comparable degrees of esterification. The approach developed enables a very detailed study of the methyl-ester distribution of pectin to be carried out and is a very important addition in the study of the functional behavior of this complex polymer.     

Short-term toxic effects of chlorobenzenes on broadbean (Vicia faba) seedlings

The root growth, changes in Superoxide dismutase (SOD, EC 1.15.1.1) activity, malonyldialdehyde (MDA) and total soluble protein level of broadbean (Vicia faba) seedlings were researched at different soil concentrations of chlorobenzene (CB), 1,2,4-trichlorobenzene (TCB) and hexachlorobenzene (HCB). The results showed that root growth of seedlings was interrupted after 5d of 50–200 μg · g⁻¹ TCB treatment. During a 3 d of recovery period, root growth was, however, restored to some extent although there was a delay in returning to the control level. The total soluble protein content in seedlings increased with TCB concentration and duration of exposure. Effect of TCB stress on SOD activity in seedlings displayed a significant dose-effect relationship for 1–5 d of 50–200 μg · g⁻¹ treatment. When broadbean seedlings were placed in clean tap water for 3 d following exposure to 5 d of TCB stress to clear tap water for 3 d, SOD activity at 50 μg · g⁻¹ TCB recovered towards control level (P> 0.05) while a significant increase in SOD activity was observed at 100 and 200 μg · g⁻¹ TCB compared to control (P< 0.05). The experiments also revealed that a significant increase of MDA level in seedlings occurred after 3 and 5 d of 100 and 200 μg · g⁻¹ TCB treatment (P< 0.05 andP< 0.01), and there was a positive correlation between TCB concentration and MDA level. All the above results showed that SOD activity and MDA level of broadbean seedlings might be proposed as the biomarkers for short-term TCB contamination in soil. Compared to TCB, the toxicity of 50−1000 μg · g⁻¹ CB or HCB in soil to broadbean seedlings was not observed after a 3 d exposure.

     

Short-term toxic effects of chlorobenzenes on broadbean (<Emphasis Type="Italic">Vicia faba</Emphasis>) seedlings

The root growth, changes in Superoxide dismutase (SOD, EC 1.15.1.1) activity, malonyldialdehyde (MDA) and total soluble protein level of broadbean (Vicia faba) seedlings were researched at different soil concentrations of chlorobenzene (CB), 1,2,4-trichlorobenzene (TCB) and hexachlorobenzene (HCB). The results showed that root growth of seedlings was interrupted after 5d of 50–200 μg · g^?1 TCB treatment. During a 3 d of recovery period, root growth was, however, restored to some extent although there was a delay in returning to the control level. The total soluble protein content in seedlings increased with TCB concentration and duration of exposure. Effect of TCB stress on SOD activity in seedlings displayed a significant dose-effect relationship for 1–5 d of 50–200 μg · g^?1 treatment. When broadbean seedlings were placed in clean tap water for 3 d following exposure to 5 d of TCB stress to clear tap water for 3 d, SOD activity at 50 μg · g^?1 TCB recovered towards control level (P> 0.05) while a significant increase in SOD activity was observed at 100 and 200 μg · g^?1 TCB compared to control (P< 0.05). The experiments also revealed that a significant increase of MDA level in seedlings occurred after 3 and 5 d of 100 and 200 μg · g^?1 TCB treatment (P< 0.05 andP< 0.01), and there was a positive correlation between TCB concentration and MDA level. All the above results showed that SOD activity and MDA level of broadbean seedlings might be proposed as the biomarkers for short-term TCB contamination in soil. Compared to TCB, the toxicity of 50?1000 μg · g^?1 CB or HCB in soil to broadbean seedlings was not observed after a 3 d exposure.     

An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR

A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and ¹³C NMR. Optimum conditions afford samples at 50–80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for ¹³C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for M_W > 4 kDa. The M_W values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples. Received: 10 July 1997 / Accepted: 12 November 1997     

Rheology of Mixed Pectin Solutions

The effects of sucrose (S) and pectin (P) concentrations and the ratio between two distinct pectins (R) on the rheological behavior of diluted pectin systems were evaluated simultaneously using the surface response methodology. The systems were composed of a mixture of two high methoxy pectins with different degree of methyl esterification values (HM1/HM2) and of a mixture of a high-methoxy with an amidated low-methoxy pectin (HM1/LMA). For the HM1/HM2 systems, the multivariate analysis showed that the sucrose and pectin concentrations exerted statistically significant (p < 0.05) linear effects on the consistency index k and viscosity, the influence of pectin being about five times higher than that of sucrose. The pectin concentration and the ratio between the different pectins were shown to be significant with respect to the rheological parameters of the HM1/LMA systems. Evaluating the influence of the ratio between the different pectins, a synergistic effect on the structure reinforcement was observed when mixing HM1 and LMA in similar proportions, indicating the importance of the presence of hydrophobic interactions between methyl ester groups in addition to the stronger hydrogen bonding in junction zone stabilization. In general, the conditions in which hydrogen bonds were favored in relation to hydrophobic interactions led to systems with higher pseudoplasticity.     

Chemical composition and construction cost for roots of Mediterranean trees, shrub species and grassland communities

The construction cost of fine roots was studied in 23 woody species and two grassland communities, growing under natural conditions in southern Spain. Calculation of the energy (glucose) required for their synthesis was based on the quantification of chemical components present in tissues. Despite considerable differences in the chemical composition of the three life forms studied (trees, shrubs and herbaceous), detected differences in construction cost were non‐significant (mean value: 1·64 ± 0·13 g glucose g^?1). However, shrubs and herbaceous plants growing in more fertile habitats expended significantly less energy on root synthesis (1·58 ± 0·06 and 1·41 ± 0·05 g glucose g^?1, respectively) than those growing in less fertile areas (1·80 ± 0·06 and 1·57 ± 0·1 g glucose g^?1, respectively), because they contained smaller amounts of either waxes (shrubs) or lignins (herbaceous), both expensive to synthesize, and, proportionately, more cellulose; which is inexpensive to synthesize. Deciduous and evergreen tree species also differed mainly with regard to wax and cellulose contents, giving rise to a significantly higher construction cost in evergreens (1·57 ± 0·07 g glucose g^?1 versus 1·78 ± 0·02 g glucose g^?1). The differences observed in construction cost appeared to be due more to habitat‐induced differences in chemical composition than to any intrinsic difference between the species studied.     

Optimization of gluconic acid synthesis by removing limitations and inhibitions

The optimization task was performed using the gluconic acid synthesis by the Acetobacter methanolicusMB 58 strain. The microorganisms were grown continuously on methanol as the growth substrate. After finishing the growth process by the deficiency of N and P, the gluconic acid synthesis was started by adding glucose. The synthesis process was performed continuously. The oxygen transfer rate depended on the gluconic acid concentration. During the growth process, the oxygen transfer rate reached a value of about 13 g O₂ · kg^?1 · h^?1using a 30-l glass fermenter equipped with a 6 blade stirrer and fully baffled. This rate declined to a value of between 2 and 5 g O₂ · kg^?1 · h^?1 in the presence of gluconic acid concentrations above 150 g gluconic acid · kg^?1medium. The yield (g gluconic acid · g^?1glucose) depended on the gluconic acid concentration and amounted to y = 0.7 in relation to 150 g gluconic acid · kg^?1medium and y = 0.8 in relation to 200 g · kg^?1medium, respectively. The fermenters were coupled with ultrafiltration moduls (Fa. ROMICON and Fa. SARTORIUS). The biomass concentrations amounted from 5 to 40 g dry mass kg^?1medium. The ultrafiltration modules retained the biomass within the fermentation system. A glucose solution (30 to 50 weight percent glucose) was continuously dosed into the fermenter. The retention time was chosen between 2 and 30 h. The gluconic acid synthesis rate reached values of up to 32 g gluconic acid · kg^?1 · h^?1. Within a range of up to 250 g gluconic acid · kg^?1medium, the acid concentration had no influence on the enzyme activity.     

Structural study of the solutions of acidic polysaccharides. II. Study of some thermodynamic properties of the dilute pectin solutions with different degrees of esterification

Light-scattering and surface tension measurements have been used to study the structure and thermodynamic properties of aqueous-salt solutions of pectins, with the degree of esterification (DE) varying from 5 to 100%. Pectin macromolecules were found in the form of aggregates with $\text{M}{}_{\mathrm{w}}\text{= 5 × 10}{}^5\text{to}\text{1·1 × 10}{}^6\text{D}$. The second virial coefficient of pectin was negative in these poor solvents and independent of the DE. Pectins exhibited surface activity at the air-water interface, the magnitude of this activity also being independent of the DE.     

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry‐heat treatment

Aims: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO₂) and dry heat in reducing the number of Escherichia coli O157:H7. Methods and Results: Radish seeds containing E. coli O157:H7 at 5·5 log CFU g^?1 were treated with 500 μg ml^?1 ClO₂ for 5 min and subsequently heated at 60°C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4·8 log CFU g^?1 after 12 h dry‐heat treatment. The pathogen was inactivated after 48 h dry‐heat treatment, but the germination rate of treated seeds was substantially reduced from 91·2 ± 5·0% to 68·7 ± 12·3%. Conclusions: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO₂ and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry‐heat treatment should be investigated, and conditions for drying and dry‐heat treatment should be optimized. Significance and Impact of the study: This study showed that sequential treatment with ClO₂ and dry‐heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.     

Pectin immunolocalization and calcium visualization in differentiating derivatives from poplar cambium

Summary Calcium distribution and pectin esterification patterns in the cambial zone of poplar branches were studied with ionic microscopy and immunological tools respectively. Dynamic changes correlating with cell growth and cell differentiation were observed both on the xylem and on the phloem sides. In expanding cell walls of xylem derivatives, unesterified pectins were restricted to cell junctions and middle lamellae, occasionally accompanied by calcium ions. In contrast, in differentiating and mature phloem cells, acidic pectins and Ca²⁺ were present all over the walls leading to early stiffening of the polysaccharide network. Significant labelling was detected with JIM5 antibodies in some dictyosomes suggesting exocytosis of low methylated polymers towards the cell walls. At cell junctions, unesterified pectins might originate from the activity of pectinmethylesterases localized in these areas. Thus un- and deesterified pectins might be located in different cell wall domains whose distribution, varying with cell type, will confer specific extensibility to the wall matrix.Abbreviations BSA bovine serum albumin - DM degree of methylation - FITC fluorescein isothiocyanate - HM highly methylated pectins - LM low methylated pectins - PME pectin methylesterase - SIMS secondary ion mass spectrometry - TBS tris-buffered saline     

Comparison of spatial variation in otolith chemistry of two fish species and relationships with water chemistry and otolith growth

Spatial variation in the chemistry (Mg, Mn, Sr and Ba) of recently deposited otolith material (last 20–30 days of life) was compared between two demersal fish species; snapper Pagrus auratus (Sparidae) and sand flathead Platycephalus bassensis (Platycephalidae), that were collected simultaneously at 12 sites across three bays in Victoria, south-eastern Australia. Otolith chemistry was also compared with ambient water chemistry and among three sampling positions adjacent to the proximal otolith margin. For both species, variation in otolith chemistry among bays was significant for Ba, Mn and Sr; however, differences among bays were only similar between species for Ba and Mn. Only Ba showed significant variation at the site level. Across the 12 sites, mean otolith Ba levels were significantly positively correlated between species. Further, although incorporation rates differed, mean ambient Ba levels for both species were positively correlated with ambient Ba levels. Spatial variation in multi-element otolith chemistry was also broadly similar between species and with multi-element water chemistry. Partition coefficients clearly indicated species-specific incorporation of elements into otoliths. Mg and Mn were consistently higher in snapper than sand flathead otoliths (mean ±s .d ., Mg snapper 22·1 ± 3·8 and sand flathead 9·9 ± 1·5 μg g⁻¹, Mn snapper 4·4 ± 2·6 and sand flathead 0·5 ± 0·3 μg g⁻¹), Sr was generally higher in sand flathead otoliths (sand flathead 1570 ± 235 and snapper 1346 ± 104 μg g⁻¹) and Ba was generally higher in snapper otoliths (snapper 12·1 ± 12·8 and sand flathead 1·8 ± 1·4 μg g⁻¹). For both species, Mg and Mn were higher in the faster accreting regions of the otolith margin, Sr was lower in the slower accreting region and Ba showed negligible variation among the three sampling regions. This pattern was consistent with the higher Mg and Mn, and generally lower Sr observed in the faster accreting snapper otoliths. It is hypothesized that the differences between species in the incorporation of these elements may be at least partly related to differences in metabolic and otolith accretion rate. Although rates of elemental incorporation into otoliths appear species specific, for elements such as Ba where incorporation appears consistently related to ambient concentrations, spatial variation in otolith chemistry should show similarity among co-occurring species.     

Uptake of glycine and release of primary amines by the polychaete Nereis virens (Sars) and the mud snail Hydrobia neglecta muus

Uptake of glycine and release of primary amines by the polychaete Nereis virens (Sars) and the mud snail Hydrobia neglecta Muus was investigated. In a flow system, Nereis virens had a net influx of glycine of 32 to 39 nmol·g^?1 fresh wt·h from a 5-μM solution of glycine. Simultaneously, an efflux of primary amines of 14 nmol·g^?1·h^?1 was measured. The experiments indicated that glycine uptake and release of primary amines are two independent processes. Glycine was absorved by Hydrobia neglecta from 50-μM solutions at an influx of 175 nmol·g^?1 organic wt·h^?1, and simultaneously, there was an independent efflux of 25 nmol primary amine·g^?1·h^?1. In the experiments, released primary amines from both species increased the ambient concentration of primary amines, but after various periods of time these concentrations apparently stabilized. Finally, the significance of micro-organisms in amino-acid uptake experiments is considered.     

The Distribution of Mercury in the Sediments of Eight Saskatchewan Lakes

Eight lakes located in the southern half of Saskatchewan were sampled in May and June, 1987, to determine the concentration and spatial distribution of mercury in their surface sediments. Katepwa and Buffalo Pound lakes had maximum total mercury sediment concentrations of 0.158 and 0.100 μg · g⁻¹ dry weight respectively, while in the other six lakes the mercury concentration was never greater than 0.04 μg · g⁻¹ dw. Despite this difference in mercury concentration in the sediments of the lakes, walleye sampled from them have high mercury concentrations exceeding 0.5 μ · g⁻¹ (wet weight).