Ethanolic fermentation with saccharomyces cerevisiae cells immobilized in pectate gel

High-molecular weight pectic acid with a STAUDINGER index of 210 ml/g and a degree of esterification of 3%was used as matrix material for the immobilization of Saccharomyces cerevisiae cells. In discontinuous and continuous fermentation tests the gel beads obtained exhibited the same biomass loading capacity (152–155 g dry wt. cells/kg gel) and about the same maximum specific productivity (103.0 g ethanol/kg gel · h) as alginate immobilizates. But there were distinct differences in the swelling behaviour of the two gels. Under the same experimental conditions the increase of bead volume amounted to 27% only for pectate gel in comparison to 129% for alginate gel. In continuous fermentation experiments performed in a horizontal-column packed-bed reactor with liquid recycling a mean steady-state ethanol concetration of 69.1 g/l and a mean productivity of 24.7 g ethanol/lh could be kept constant over a period of more than 10 days.     

Physicochemical behaviour of chitin gels

Syneresis of chitin gels formed in the course of N-acetylation of chitosan in hydroalcoholic media has been studied. A critical cross-linking density related to a critical acetylation degree for which the gel undergoes weak syneresis and swells in water was shown (degree of acetylation (DA) 88%). Above this value, the weight loss during syneresis increases with DA. Conversely, syneresis decreases on increasing the polymer concentration, but disappears at a macroscopic level for a polymer concentration close to the critical concentration of entanglement in the initial solution. An increase in temperature favours the formation of hydrophobic interactions and new inter- and intramolecular hydrogen bondings. Due to the weak polyelectrolyte character of chitin, the weight of the gel depends on the pH and ionic strength of the media. Swelling-deswelling experiments show that the swelling of the gel is not fully reversible in relation with the formation of new cross-links during the depletion of the network. Our results reveals that the balance between segment-segment and segment-solvent interactions as well as the molecular mobility play the major role.     

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.     

Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical characterization of solvent-induced systems

Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.     

Development of a Model for Analyzing the Swelling Rate of Ionic Gels on the Basis of the Diffusion of Mobile Ions: Application to the pH-Sensitive Swelling of a Polyelectrolyte Complex Gel Prepared from Xanthan and Chitosan

A model for analyzing the swelling rate of ionic gels was developed on the basis of the diffusion of a species of mobile ion. This model was applied to the analysis of pH-sensitive swelling of a xanthan/chitosan complex gel in NaOH solutions of pH 9–12, using the sodium ion as the reference mobile ion. The time–course for swelling of gel beads with a pH change from 11 to 10 was successfully described by the developed model. The values for the diffusion coefficient obtained by fitting the model to the data were of the same order as those for the diffusion coefficient of the sodium ion measured for a membrane of the complex gel. Thus, it was confirmed that the swelling rate of the gel due to pH change was mainly controlled by the diffusion of mobile ions. However, the time-course for swelling of the gel at pH values below 10 was not satisfactorily explained by the model developed, suggesting that the change in the degree of ionization during swelling also affected the swelling rate of the xanthan/chitosan complex gel.     

Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Chitin gels

Chitin dissolved in N,N-dimethylacetamide, N-methyl-2-pyrrolidone and their mixed solvents in the presence of 5% LiCl was treated with acetic anhydride-pyridine, and the mixture solution was heated at 100 degrees C for 6 h to give a partially O-acetylated chitin gel. Chitin dissolved in these solvents in the presence of 5% LiCl was mixed with pyridine, and the mixture solution was heated at 60 degrees C for 5 h to give a chitin gel. Both the gels were rigid and transparent, and their properties and the rate of the hydrolysis of the chitin xerogel by hen-egg white lysozyme were essentially similar to those of N-acetylchitosan gel prepared by chemical N-acetylation of chitosan.     

Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels

In order to optimize the use of proteins as functional ingredients in foods, one needs more insight into the effects of environmental conditions (pH, ionic strength, and temperature) on the functional properties of protein. This paper summarizes the results of an extensive study on heat-induced gelation of ovalbumin (egg-white protein) and soybean protein in the concentration range from 10 to 35 g/100 g. It was the aim of the study to relate the rheological properties of thermally induced protein gels to the microstructure of the gel and the physicochemical properties of the constituent protein. The gelling behavior of the protein was quantified with rheological techniques, and the physical properties of the gels were determined, at small and large deformations. From the swelling/dissolving behavior of the gels in various media, the nature of the crosslinks was determined qualitatively. The microstructure of the gels was determined with electron microscopy. Nmr-spectroscopy was applied in order to elucidate changes in conformation during heating. It was found that the formation of a continuous covalently crosslinked network is not a prerequisite for thermally-induced protein gelation. The properties of a gel strongly depend on the pH at which the gel is formed. When heat-set at high pH(pH～10), a homogeneous, strong, and almost transparent gel is formed, consisting of flexible crosslinked protein gels. Heat-setting at low pH (pH 5) leads to the formation of a heterogeneous and weak gel, which easily exudes water. This gel consists of crosslinked aggregated protein. The ionic strength of the solvent in which the protein is dissolved and heat-set has a much lower effect on gel properties.     

Swelling behavior and controlled release of theophylline and sulfamethoxazole drugs in beta-lactoglobulin protein gels obtained by phase separation in water/ethanol mixture

Physically cross-linked beta-lactoglobulin (BLG) protein gels containing theophylline and sulfamethoxazole low molecular weight drugs were prepared in 50% ethanol solution at pH 8 and two protein concentrations (6 and 7% (w/v)). Swelling behavior of cylindrical gels showed that, irrespective of the hydrated or dehydrated state of the gel, the rate of swelling was the highest in water. When the gels were exposed to water, they first showed a swelling phase in which their weight increased 3 and 30 times for hydrated and dehydrated gels, respectively, due to absorption of water, followed by a dissolution phase. The absorption of solvent was however considerably reduced when the gels were exposed to aqueous buffer solutions. The release behavior of both theophylline and sulfamethoxazole drugs from BLG gels was achieved in a time window ranging from 6 to 24 h. The drug release depended mainly on the solubility of the drugs and the physical state of the gel (hydrated or dry form). Analysis of drug release profiles using the model of Peppas showed that diffusion through hydrated gels was governed by a Fickian process whereas diffusion through dehydrated gels was governed partly by the swelling capacities of the gel but also by the structural rearrangements inside the network occurring during dehydration step. By a judicious selection of protein concentration, hydrated or dehydrated gel state, drug release may be modulated to be engineered suitable for pharmaceutical as well as cosmetics and food applications.     

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma

Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m(2)/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 microM/g of polymer. Then, Cu(2+) ions were chelated on the beads. Cu(2+)-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu(2+)-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption-desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.     

Improving immobilized biocatalysts by gel phase polymerization

A new method is presented for the treatment of gel-type supports, used for immobilizing microbial cells and enzymes, to obtain high mechanical strength. It is particularly useful for ethanol fermentation over gel beads containing immobilized viable cells, where the beads can be ruptured by gas production and the growth of cells within the gels. This method consists of treating agar or carrageenan gel with polyacrylamide to form a rigid support which retains the high catalytic activity characteristic of the untreated biocatalysts. The size and shape of the biocatalyst is unaffected by this treatment. The method involves the diffusion of acrylamide, N,N'-methylenebisacrylamide and beta-dimethylaminopropionitrile (or N,N,N',N'-tetramethyl-ethylenediamine) into the performed biocatalyst beads followed by the addition of an initiator to cause polymerization within the beads. Treated gels have been used for the continuous fermentation of glucose to ethanol in a packed column for over two months. During this operation, the gel beads maintained their rigidity, and the maximum productivity was as high as 50 g h(-1) L(-1) gel. There was no appreciable decay of cell activity.     

Stability of alginate-immobilized algal cells

Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead characteristics were found to be affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation was found to involve a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable calcium alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5muM. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.     

Swelling and mechanical properties of biopolymer hydrogels containing chitosan and bovine serum albumin

Experimental and theoretical investigations of the swelling and mechanical properties of hydrogels formed from chitosan, bovine serum albumin (BSA), and chitosan/BSA mixtures cross-linked with genipin were performed. The properties of cross-linked chitosan hydrogels were explained in terms of its polyelectrolyte behavior, which led to a gradual increase in swelling ratio below the pK value, but whereby its swelling ability was eliminated by the presence of salt that screened the charges. Comparison of theoretical and experimental calculations of the swelling ratio, however, indicated that complications arising from wastage of cross-links, and formation of polymerized genipin cross-links must be considered before quantitative prediction can be achieved. Cross-linked BSA hydrogels swelled even in the presence of salt, and a marked increase in swelling was observed below pH = 3 that was explained as the result of an acid induced denaturation of the protein that led to unfolding of the molecule. Swollen BSA hydrogels were mechanically weak, however. Composite gels made from a cross-linked mixture of chitosan and BSA exhibited the swelling behavior of BSA combined with the mechanical properties of chitosan and were therefore considered most suitable for use in a gastric environment.     

Elaboration and characterization of whey protein beads by an emulsification/cold gelation process: application for the protection of retinol

Whey protein beads were successfully produced using a new emulsification/cold gelation method. The principle of this method is based on an emulsifying step followed by a Ca(2+)-induced gelation of pre-denatured (80 degreesC/30 min) whey protein. Beads are formed by the dropwise addition of the suspension into a calcium chloride (CaCl(2)) solution. IR results show that bead formation has a pronounced effect on the secondary structure of whey protein, which leads to the formation of intermolecular hydrogen-bonded beta-sheet structures. Their preparation conditions (CaCl(2) concentrations of 10, 15, and 20% (w/w)) influence their sphericity and homogeneity: an increase in CaCl(2) favors regular-shaped beads. The physicochemical and mechanical characterizations of beads were also carried out. Their properties, such as swelling, elasticity, deformability, and resistance at fracture, change according to pH levels (1.9, 4.5, and 7.5) and preparation conditions. Indeed, protein chain networks exhibit different behavior patterns with respect to their charge. Finally, bead degradation by enzymatic hydrolysis reveals that beads are gastroresistant and form good matrixes to protect fat-soluble bioactive molecules such as retinol, that have in vivo intestinal absorption sites. The experiment demonstrated the potential of whey protein beads to protect molecules sensitive (i.e., vitamins) to oxidation.     

Native fibrin gel networks observed by 3D microscopy, permeation and turbidity

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.     

Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent

Controlled osmotic swelling and de-swelling measurements have been performed on gelatin, a polyampholyte, hydrogels suspended in water-ethanol marginal solvent at room temperature (20 degrees C) where the alcohol concentration was changed from 0 to 100% (v/v). The change in gel mass was monitored as function of time until osmotic equilibrium was established with the surrounding solvent. It was observed that osmotic pressure of polymer-solvent mixing, pi(m)相似文献   

Structural investigation of beta-lactoglobulin gelation in ethanol/water solutions.

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in hydro-ethanolic solutions (50/50% (v/v)) at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week depending on the physico-chemical conditions. The aggregation kinetics, studied by infrared spectroscopy and dynamical rheological measurements, highly depended upon the pH; the process being the fastest at pH 7. Alcohol-induced aggregation of BLG was characterized by the formation of intermolecular hydrogen bonded beta-sheet structures. Small angle neutron scattering indicated that the aggregates structures in the final gels were similar at pH 7, 8 and 9. Through the data obtained at the molecular and macroscopic levels, it can be concluded that the kinetics of gelation were pH dependent while the spatial arrangements of the aggregates were similar in the final structures. The heterogeneous structures formed in hydro-ethanolic gels could be analysed in terms of a phase separation, the syneresis being the final visible state.     

Kinetics of ethanol production by immobilized cells of Zymomonas mobilis at varying d-glucose concentrations

Ethanol fermentation by cells of Zymomonas mobilis immobilized in calcium alginate gel has been studied using 5 to 30 wt% initial d-glucose in the medium. Up to 27% d-glucose was completely fermented and the maximum ethanol concentration of 12.6% (w/v) was obtained using an immobilized cell concentration of 58 g dry wt l^?1 of bead volume. The ethanol yield coefficient was almost unaffected by initial d-glucose concentration and its value was >95% of theoretical. The rates of ethanol production and d-glucose utilization first increased, with an increase in initial d-glucose concentration up to 13.6%, and then started to decrease upon a further increase in initial d-glucose concentration. Cell leakage from the calcium alginate beads was very low.