Calcium Pectate Gel Research Articles

Extraction and characterization of pectin methylesterase from muskmelon biowaste for pectin remodeling.

Pectin methylesterase (PME) extracted from muskmelon was purified by anion exchange chromatography. The specific activity of purified enzyme was 152.01U/mg and relative molecular weight was ~69,000Da. Methylesterase was characterized for various physicochemical factors to designate its suitability in the food industry applications. The optimum temperature of the enzyme was 30°C and is thermally stable between the temperature ranges of 15-65°C with critical temperature for stability being >65°C. Thermal inactivation first order kinetics and thermodynamic parameters in temperature range (45-65°C) favors stability of PME and at 75°C complete inactivation of enzyme was observed indicating the unstable nature of enzyme over >65°C. Activation energy (Ea ) and Z values of thermal inactivation were found to be 100.108kJ/mol and 2.05°C, respectively. About 0.1M NaCl is essential for enzyme to attain the maximum activity. The enzyme lost activity in presence of divalent calcium (Ca2+ ) and magnesium (Mg2+ ) ions. PRACTICAL APPLICATIONS: Pectin methylesterase (EC3.1.1.11) are an important class of enzymes expressed in plants and microbes and they bring about the de-methylesterification on pectin substrate. Up to ~13% degree of esterification of pectin was observed with muskmelon PME enzyme treatment. The de-methylesterified pectin thus prepared was subjected for gelation in presence of Ca2+ ions and above 0.5% of demethylesterified pectin stable calcium pectate gels were produced. The study demonstrates the suitability of muskmelon PME extracted from biowaste in food applications with good gelling property.

Dissolution kinetics of polyphenol bearing calcium pectate hydrogels in simulated gastric or intestinal media and their anti-carcinogenic capacities

Cherry laurel is an underutilized agricultural resource with a significant content of polyphenolic compounds that potentially may demonstrate antioxidant and anti-carcinogenic activities. In this study, the preparation of calcium pectate gels bearing cherry laurel polyphenols was aimed and their dissolution behaviors in simulated gastric and intestinal media were tested. In addition, the released fractions were collected and tested in cell culture media in order to determine the anti-proliferative characteristics. Cherry laurel polyphenol extracts were mixed with pectin dispersions (1:1) and calcium pectate gels were prepared by the extrusion dripping method. The freeze dried gel samples were analyzed for morphological characteristics (SEM) and dissolution kinetics in simulated gastrointestinal media. The rate of release in gastric medium was substantial (≥94.9%) in all cases and dissolved fractions demonstrated significant anti-proliferative activity (approx. 85% inhibition for 55K06 and 61K04 extracts) against MCT 116 colon carcinoma cells even after 20× dilution. The findings were discussed with emphasis on their relevance to pectin-polyphenol interactions in dissolution behavior of the gels in simulated gastrointestinal media, and also to the preparation of functional lyophilizates that can be utilized in functional foods and pharmaceuticals.

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene- co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene- co-guanidine) (PMCG), CaCl 2 and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.

Applicability of pectate-entrapped Lactobacillus casei cells for l(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of L: (+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce L: (+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.

Efficiency of a fixed-bed and a gas-lift three-column reactor for continuous production of ethanol by pectate-and alginate-immobilized Saccharomyces cerevisiae cells

AbstractEthanol-tolerant and thermo-tolerant yeast strain Saccharomyces cerevisiae C11-3 cells immobilized in calcium pectate and calcium alginate gels were used for ethanol fermentation in a three-reactor system with a gradient temperature control. The fermentation process has been tested in a fixed-bed and a gas-lift arrangement. The gas-lift system was more efficient due to a better mass transport between the phases. Abrasion was more evident in calcium alginate particles, while calcium pectate beads were not significantly damaged. Two different concentrations of alginate were tested and calcium pectate gel was demonstrated to be more suitable as an immobilization material in comparison with calcium alginate due to its mechanical resistance and favourable diffusion parameters, providing an ethanol production of more than 7.5 g dm−3 h−1 over a period of 630 h.

Flow calorimetry--a useful tool for determination of immobilized cis-epoxysuccinate hydrolase activity from Nocardia tartaricans.

Bacterial cells Nocardia tartaricans with cis-epoxysuccinate hydrolase activity were entrapped in hardened calcium pectate gel by a commercial high performance encapsulator. This enzyme (in a single step reaction with no formation of side products) was used to hydrolyze disodium cis-epoxysuccinate to a pure enantiomer--disodium L-(+)-tartrate. Activities of this enzyme were determined using flow calorimetry. The validity of this method was corroborated by HPLC and isotachophoresis. The immobilized biocatalyst has activity (75.8 U/mgdry) able to convert disodium cis-epoxysuccinate to disodium tartrate at 94% yield in 5.5h. Immobilization of N. tartaricans in hardened calcium pectate gel beads had a positive effect on the activity of cis-epoxysuccinate hydrolase, storage stability, yield, and time of bioconversion.

A role for pectin in the control of cell expansion

Uptake of nutrients and water depends on the growth of roots through elongation of individual cells near the root tip. Many of the numerous components of Type I primary cell walls, those of dicotyledons and monocotyledons other than grasses (Poaceae), have been determined, and many hypotheses have been proposed for the control of cell expansion. This important aspect of plant growth still needs elucidation, however. A model is proposed in which pectin, which occurs as a calcium (Ca) pectate gel between the load-bearing cellulose microfibrils and xyloglucan (XG) chains, controls the rate at which cells expand. It is considered that the increasing tension generated by the expanding cell is transmitted to interlocked XG chains and cellulose microfibrils. The resulting deformation of the embedded Ca pectate gel elicits the excretion of protons from the cytoplasm, possibly via compounds such as cell wall-associated kinases, that weakens the Ca pectate gel, permitting slippage of XG molecules through the action of expansin. Further slippage is prevented by deformation of the pectic gel, proton diffusion, and the transfer of residual tension to adjacent XG chains. Evidence for this model is based on the effects of pH, Ca, and aluminum (Al) on root elongation and on the reactions of these cations with Ca pectate. This model allows for genetic selection of plants and adaptation of individual plants to root environmental conditions.

Cytological Characterization of Elicitin-Induced Protection in Tobacco Plants Infected by Phytophthora parasitica or Phytoplasma

ABSTRACT Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultrastructural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloem-restricted microorganisms.

Nucleoside triphosphates production using recombinant Escherichia coli entrapped in calcium pectate gel

Nucleoside triphosphates, important intermediates in oligosaccharide synthesis by glycosyltransferases, were generated from nucleoside monophosphates by E. coli BL21(DE3) which over-expresses polyphosphate kinase (PPK) or by Corynebacterium ammoniagenes ATCC 21264 using 1% (w/v) polyphosphate as a phosphate donor and a source of energy. Beads of calcium pectate gel were stable in polyphosphate reaction broth for 60 days after which the activity of PPK was 50% of its original value. This technique could be used for the large-scale synthesis of oligosaccharides.

Monitoring of the bioconversion of glycerol to dihydroxyacetone with immobilized Gluconobacter oxydans cell using thermometric flow injection analysis

The bioconversion of glycerol to dihydroxyacetone (DHA) by Gluconobacter oxydans cells was monitored by flow microcalorimetry. DHA was produced in an air-lift reactor using bacterial cells immobilized in calcium pectate gel. Glycerokinase and galactose oxidase were immobilized on controlled pore glass and used for the determination of glycerol (substrate) and dihydroxyacetone (product), respectively. Concentration values obtained by flow microcalorimetry were in a good correlation with a spectrophotometric reference method. Furthermore, flow microcalorimetry can be used for on-line monitoring of the process.

Batch and continuous mead production with pectate immobilised, ethanol-tolerant yeast

Saccharomyces cerevisiae immobilised in calcium pectate gel optimally fermented honey mash to mead with a beads/medium ratio of 1:3, at 30 °C with `Vitamon Ultra' salt as the best commercial nitrogen/nutrient source. In continuous fermentation using a two-column system, the overall ethanol production rate was 5.7 g l−1 h−1.

The fungal elicitor cryptogein induces cell wall modifications on tobacco cell suspension.

Upon addition of the fungal elicitor cryptogein, suspension cells of tobacco (Nicotiana tabacum cv. Xanthi) aggregated in clusters. Cytochemical experiments indicated that elicited cells displayed fibrillar expansions of pectin along the primary cell wall. Immunocytochemical detection of pectin epitopes indicated that the fibrillar material surrounding the treated cells was mostly composed of low methylated galacturonan sequences, but the use of the cationic probe did not reveal the presence of negatively charged carboxyl groups: the presence of important amounts of calcium ions in these pectic fibrillar expansions accounts for these observations. These data indicate that tobacco cells treated with cryptogein show a cell wall altered by the presence of a calcium pectate gel, resulting from the reorganization of pectin in the middle lamellae. These results are consistent with a drastic reduction in wall digestibility, partially reversed by increasing the pectolyase concentration in the hydrolytic solution. Diphenylene iodonium, an inhibitor of the oxidative burst triggered by cryptogein on tobacco cells, partially prevents elicited cell walls from this loss of digestibility, suggesting a possible role of active oxygen species in the cell wall strengthening. This work represents a new element of the signal transduction cascade triggered on tobacco cells by cryptogein.

Calcium pectate gel beads for cell entrapment. 6. Morphology of stabilized and hardened calcium pectate gel beads with cells for immobilized biotechnology

The structure of standard and stabilized calcium pectate gel (CPG) beads has been examined by scanning (SEM) and transmission (TEM) electron microscopy. A two-stage crosslinking procedure with polyethyleneimine (PEI) and glutaraldehyde (GA) led to the formation of a more compact layer on the bead surface. On the other hand, the stabilization procedure did not significantly change either gel bead interior or morphologic properties, vitality and biotransformation activity of immobilized bacterial cells (Nocardia tartaricans) against cis-epoxysuccinate as well as yeast cells (Trigonopsis variabilis) against cephalosporin C. The structure of these cells within the calcium pectate matrix remained unchanged. Moreover, the two-step chemical stabilization of CPG containing T. variabilis or N. tartaricans had a favourable effect on storage and operational stability at semi-continuous and continuous processing in stirred batch and packed-bed reactors. The most valuable effect of stabilization was the fact that the hardened CPG comprising the cells N. tartaricans resisted, for a long time (360 days and more), the destructive effects of the product (such strong sequestering reagent as L-(+)-tartaric acid) at high concentrations (up to 1M). Non-hardened CPG was destroyed after 21 h. The reference materials, hardened and non-hardened calcium alginate gels (CAG), were destroyed over 3h or 30 min, respectively.

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.

Enzyme flow microcalorimetry—a useful tool for screening of immobilized penicillin G acylase

Screening of a representative series of immobilized penicillin G acylase biocatalysts (enzyme, cells) using enzyme Now microcalorimetry is described. Immobilized penicillin G acylase biocatalysts were either prepared in the labor- atory by various techniques or obtained from four commercial manufacturers. An industrial strain of Escherichia coli was entrapped in (poly)acrylamide gel or hardened calcium pectate gel. Semi-puri-ed enzyme was immobilized in various waysEeither by covalent binding to oxirane-acrylic beads or chlorotriazine bead cellulose or by entrapment in (poly)acrylamide gel. The validity of the enzyme Now microcalorimetry results was corroborated by a pH-stat method, showing enzyme Now microcalorimetry to be a suitable method for rapid screening of immobilized biocatalysts regardless of the immobilization technique, carrier type or the biocatalyst source. 1998 Society of Chemical Industry ( J. Chem. T echnol. Biotechnol. 73 ,3 1 E36 (1998)

Comparison of ethanol tolerance of free and immobilizedSaccharomyces uvarum yeasts

O2 consumption and CO2 production of free and immobilizedSaccharomyces uvarum in the presence of ethanol were compared. The protective effect of immobilization on the yeast ethanol tolerance at 5–20% of ethanol was more evident in CO2 production than in O2 consumption. CO2 production by the yeast immobilized in calcium alginate and calcium pectate gel beads was approximately 2.5-times higher than by the free yeast at 5 and 10% of ethanol. 4-Fold increase of CO2 production was observed at 15% ethanol. Immobilization in calcium-containing carriers (alginate, pectate) resulted in enhanced activities of yeasts compared to the κ-carrageenan carrier.

Chain conformation in concentrated pectic gels: evidence from 13C NMR

13C NMR spectroscopy is a sensitive method of exploring the conformation of polysaccharides in both solid and gel states, because the positions of the peaks for the two carbons on either side of the glycosidic linkage, C-1 and C-4 for pectins, depend on the chain conformation. This approach was used to determine the conformation of calcium pectate at gel concentrations of ca. 0.3 g cm −3, comparable with those found in vivo. The solid acid, methyl-esterified, and sodium forms of pectate, which are known to have a right-handed, threefold helical ( 3 1 ) conformation, were used to define the spectral features of this conformation. These features were also present in both gel and solid calcium pectate, and were more conspicuous in the solid form. When dilute calcium pectate gels are dried they are known from CD and EXAFS evidence to undergo a conformational transition from the “egg-box” twofold helical ( 2 1 ) conformation to the 3 1 conformation. Calcium guluronate does not undergo this change, nor does calcium pectate in the presence of excess of monovalent cations. Changes in the 13C NMR spectrum marking this transition were observed on drying calcium pectate gels, were prevented by excess of K +, and were not found for guluronate. This enabled the spectral features corresponding to the 2 1 conformation to be identified. The spectral assignments were in agreement with the stereoelectronic theory of conformation-dependent chemical shifts, which also allowed a third set of peaks to be assigned to conformations dispersed between the 3 1 and 2 1 helices. About 70% of the pectate was visible in the gel spectra, the remaining chains being too mobile to be detected. It was concluded that the egg-box form, as dimers or larger aggregates, was the largest single component of the gels studied here, but that substantial quantities of 3 1 and intermediate helical aggregated forms were also present. Intact, pectin-containing plant cell walls also show spectral features characteristic of these components.

Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst

Flow microcalorimeter was used for the study of microkinetic properties of Escherichia coli cells enriched with the penicillin G acylase activity immobilized in calcium pectate gel. The experimental kinetic data were obtained by measurement of the thermometric signal in the microcalorimetric column with immobilized enzyme and described by the introduced mathematical model involving the mass transfer and reaction kinetic phenomena.

Calcium uptake and efflux, ion leakage, internal air space and cation exchange capacity in relation to mealiness in nectarine tissue

Abstract Cool stored nectarine (Prunus persica) fruit (6 weeks at 0°C) ripen abnormally and develop a dry, mealy texture with low levels of extractable juice. Effects of mealiness on calcium uptake and efflux, cation leakage and internal air space of the tissue as well as the cation exchange capacity of the cell wall material have been investigated. Mealy fruit tissue had higher rates of calcium uptake and efflux, internal air space and cation exchange capacity than that from normally ripened fruit. In both normally ripened and mealy nectarines, ion leakage increased during the ripening period, although leakage of individual cations showed differences between ripe and mealy tissue. It is suggested that differences between properties of normally ripened and mealy fruit are due to changes in the cell wall character and can be expressed in terms of calcium binding and retention properties possibly involving the binding of water into the cell wall in a calcium pectate gel.

Direct determination of the cephalosporin transforming activity of immobilized cells with use of an enzyme thermistor. 1. Verification of the mathematical model

A simple method for the direct measurement of the catalytic properties of immobilized cells in the flow minicalorimeter, the enzyme thermistor (ET), is presented. A Trigonopsis variabilis strain with cephalosporin C-transforming activity was used as the model system. The yeast cells were immobilized either by crosslinking with a homobifunctional reagent or by entrapment in gels. The actual activity of the immobilized cells used in the ET was estimated by means of a stirred-batch reactor measurement in conjunction with HPLC analysis of substrate and products. Similar results were also obtained using d- amino acid oxidase (EC 1.4.3.3) isolated from T. variabilis cells and immobilized by gel entrapment. This calibration procedure was found to be appropriate for all biocatalyst systems used. The thermometric signal was proportional to the amount of biocatalyst immobilized in the ET minicolumn. It was shown that the rate of reaction catalyzed by T. variabilis entrapped in calcium pectate gel was limited by internal diffusion to an extent depending on the cell concentration in the biocatalyst particle. This approach offers a direct method for studying the kinetic properties of immobilized cells.