Enzymatic protein hydrolysis in a membrane reactor related to taste properties

Potato, Vicia faba and soybean proteins were hydrolysed enzymatically in a substrate feed membrane reactor system. Alkaline proteolytic enzymes and PM-10 membranes were used for the hydrolysis of potato protein. The taste of the ultrafiltrates, which was unpleasantly bitter and potato-like, was improved by application of gelatin. Also using PM-10 membranes, Vicia faba protein isolate was hydrolysed by alkaline and acid proteolytic enzymes. The bitterness of the ultrafiltrate decreased with the formation of an isoelectric precipitate, which was probably due to association of hydrophobic peptides. The reactor equipped with a cellulose acetate membrane delivered an acceptable enzymatic hydrolysate of Promine D during the first hours of ultrafiltration. This was not the case when similar processes were performed using non-cellulosic DM-5 membranes. The usefulness of ultrafiltration for obtaining bland protein hydrolysates seems to be limited to short-term processes with cellulose acetate membranes.     

Unique gelation behavior of cellulose in NaOH/urea aqueous solution

A transparent cellulose solution was prepared by mixing 7 wt % NaOH with 12 wt % urea aqueous solution which was precooled to below -10 degrees C and which was able to rapidly dissolve cellulose at ambient temperature. The rheological properties and behavior of the gel-formed cellulose solution were investigated by using dynamic viscoelastic measurement. The effects of temperature, time, cellulose molecular weight, and concentrations on both the shear storage modulus (G') and the loss modulus (G") were analyzed. The cellulose solution having a viscosity-average molecular weight (M(eta)) of 11.4 x 10(4) had its sol-gel transition temperature decreased from 60.3 to 30.5 degrees C with an increase of its concentration from 3 to 5 wt %. The gelation temperature of a 4 wt % cellulose solution dropped from 59.4 to 30.5 degrees C as the M(eta) value was increased from 4.5 x 10(4) to 11.4 x 10(4). Interestingly, at either higher temperature (above 30 degrees C), or lower temperature (below -3 degrees C), or for longer gelation time, gels could form in the cellulose solutions. However, the cellulose solution remains a liquid state for a long time at the temperature range from 0 to 5 degrees C. For the first time, we revealed an irreversible gelation in the cellulose solution system. The gel having been formed did not dissolve even when cooled to the temperature of -10 degrees C, at which it was dissolved previously. Therefore, this indicates that either heating or cooling treatment could not break such stable gels. A high apparent activation energy (E(a)) of the cellulose solution below 0 degrees C was obtained and was used to explain the gel formation under the cooling process.     

Preparation of levoglucosenone through sulfuric acid promoted pyrolysis of bagasse at low temperature

Fast pyrolysis of bagasse pretreated by sulfuric acid was conducted in a fixed bed reactor to prepare levoglucosenone (LGO), a very important anhydrosugar for organic synthesis. The liquid yield and LGO yield were studied at temperatures from 240 to 350 °C and sulfuric acid loadings from 0.92 to 7.10 wt.%. An optimal LGO yield of 7.58 wt.% was obtained at 270 °C with a sulfuric acid pretreatment concentration of 0.05 M (corresponding to 4.28 wt.% sulfuric acid loading). For comparison, microcrystalline cellulose pretreated by 0.05 M sulfuric acid solution was pyrolyzed at temperature from 270 °C to 320 °C, and bagasse loaded with 3-5 wt.% phosphoric acid was pyrolyzed at temperature from 270 °C to 350 °C. The highest yield of LGO from bagasse was 30% higher than that from microcrystalline cellulose, and treatment with sulfuric acid allowed a 21% higher yield than treatment with phosphoric acid.     

Cellodextrin preparation by mixed-acid hydrolysis and chromatographic separation

A procedure for preparation of purified cellodextrins in gram quantities was developed for use in biochemical and microbiological studies. Cellodextrins were prepared by hydrolyzing microcrystalline cellulose (Avicel) over a period of 4 to 5.5h in the presence of a mixture of 80% (v/v) concentrated hydrochloric acid ( approximately 37 wt.%) and 20% (v/v) concentrated sulfuric acid ( approximately 98 wt.%) at room temperature (22 degrees C). Acetone precipitation, washing ion exchange, and neutralization with barium hydroxide were used to generate a solution of mixed cellodextrins substantially free of acids and salts. Yields following hydrolysis and precipitation were approximately 0.05, approximately 0.07, approximately 0.06, and approximately 0.02 g/g cellulose for cellotriose (G(3)), cellotetraose (G(4)), cellopentose (G(5)), and cellohexose (G(6)), respectively. Cellodextrins with degrees of polymerization from 3 to 11 were separated chromatographically using a 29 x 5-cm I.D. Bio-Rad AG50W-X4 column arranged in series with a 91 x 5-cm I.D. Bio-Gel P4 column. This two-column system was used to obtain cellodextrin preparations at 240 mg/day for G(3), 330 mg/day for G(4), 260 mg/day for G(5), and 130 mg/day for G(6), with purity >99% for G(3), G(4), and G(5) and >95% for G(6). The overall procedure achieves yields comparable to the highest previously reported, employs a separation system that can readily be reused for multiple runs, and avoids use of fuming HCl.     

Optical resolution and mechanism using enantioselective cellulose,sodium alginate and hydroxypropyl‐β‐cyclodextrin membranes

Chiral solid membranes of cellulose, sodium alginate, and hydroxypropyl‐β‐cyclodextrin were prepared for chiral dialysis separations. After optimizing the membrane material concentrations, the membrane preparation conditions and the feed concentrations, enantiomeric excesses of 89.1%, 42.6%, and 59.1% were obtained for mandelic acid on the cellulose membrane, p ‐hydroxy phenylglycine on the sodium alginate membrane, and p ‐hydroxy phenylglycine on the hydroxypropyl‐β‐cyclodextrin membrane, respectively. To study the optical resolution mechanism, chiral discrimination by membrane adsorption, solid phase extraction, membrane chromatography, high‐pressure liquid chromatography ultrafiltration were performed. All of the experimental results showed that the first adsorbed enantiomer was not the enantiomer that first permeated the membrane. The crystal structures of mandelic acid and p ‐hydroxy phenylglycine are the racematic compounds. We suggest that the chiral separation mechanism of the solid membrane is “adsorption – association – diffusion,” which is able to explain the optical resolution of the enantioselective membrane. This is also the first report in which solid membranes of sodium alginate and hydroxypropyl‐β‐cyclodextrin were used in the chiral separation of p ‐hydroxy phenylglycine.     

Lipase-catalyzed cellulose acetylation in aqueous and organic media

Screening for lipases capable of catalyzing acetylation of cellulosic substrates was conducted in aqueous buffer solution using water-soluble carboxymethyl cellulose (CMC) as substrate. Lipase A12 from Aspergillus niger (A. niger) showed the most promising acetylation activity among 11 tested commercial microbial lipases and was further applied to catalyzing acetylation of solid cellulose in aqueous solution. This reaction was shown to be feasible with an acetylation extent of 0.16 wt % achieved compared with no detectable acetylation in the absence of enzyme. Pretreatments on cellulose substrate by ultrasonic irradiation and surfactant solution only slightly improved the acetylation extent by 44 and 27%, respectively. Alternatively, this lipase-catalyzed acetylation was remarkably improved with solubilized cellulose as substrate in the dimethyl sulfoxide/paraformaldehyde solvent system, with an acetylation extent (7.87 wt %) nearly 50 times higher than that achieved in aqueous solution. This improvement was attributed to (1) the absence of bulk water and the increase in substrate solubility by the transition of reaction media from aqueous solution to organic solvents and (2) the ability of lipase A12 to remain catalytically active in highly polar DMSO. This discovery that the A. niger lipase was capable of surviving its contact with polar solvents was further confirmed by its considerably preserved catalytic activity on CMC acetylation in aqueous media after enzyme pretreatments with organic solvents of various polarities and in mixture media with the aqueous phase partially replaced by organic solvents.     

Removal of organics and ions from wastewater using a hybrid system of microfiltration membrane and microorganisms

Microfiltration membranes are not able to remove organic compounds. A hybrid system of a microfiltration membrane and bacteria was designed for separation of organic compounds and ions from wastewater. Colonies of bacteria (Escherichia coli) were subjected to sedimentation on the surface of a microfiltration membrane (0.2 μm cellulose acetate) as a complementary part of the system to enhance the removal efficiency. Three selected categories of materials i.e. preservative substances, cephalosporins and ions were used to prepare the synthetic feed. The results indicate that preservatives were reduced more than 80%, cephalosporins were removed around 60% and ions decreased more than 50% in the feed solution using this hybrid system for filtration. The interaction between bacteria and chemical materials is responsible for removal of organic compounds and ions from test solutions. However the interaction decreased over time due to the limited capacity of the bacteria.     

Structure and properties of hydroxyapatite/cellulose nanocomposite films

The aim of this study was to develop a new inorganic-organic hybrid film. Nanohydroxyapaptite (nHAP) particles as the inorganic phase was mixed with cellulose in 7 wt.% NaOH/12 wt.% urea aqueous solution with cooling to prepare a blend solution, and then inorganic-organic hybrid films were fabricated by coagulating with Na₂SO₄ aqueous solution. The structure and properties of the hybrid films were characterized by high resolution transmitting electron microscopy (HRTEM), field emission scanning electron microscopy (FESEM), thermo-gravimetric analysis (TGA), Fourier transform infra-red (FT-IR) spectra, wide angle X-ray diffraction (WAXD) and tensile testing. The results revealed that the HAP nanoparticles with mean diameter of about 30 nm were uniformly dispersed and well immobilized in the hybrid film as a result of the role of the nano-and micropores in the cellulose substrate. A strong interaction existed between HAP and cellulose matrix, and their thermal stability and mechanical strength were improved as a result of good miscibility. Furthermore, the results of 293T cell viability assay indicated that the HAP/cellulose films had excellent biocompatibility and safety, showing potential applications in biomaterials.     

Dynamic viscoelastic properties of cellulose carbamate dissolved in NaOH aqueous solution

Dynamic viscoelastic properties of cellulose carbamate (CC) dissolved in NaOH aqueous solution were systematically studied for the first time. CC was microwave-assisted synthesized from the mixture of cellulose and urea and then dissolved in 7 wt % NaOH aqueous solution precooled to -7 °C. The obtained CC solution is transparent and has good liquidity. To clarify the rheological behavior of the solution, the CC solutions were investigated by dynamic viscoelastic measurements. The shear storage modulus (G') and loss modulus (G') as a function of the angular frequency (ω), concentration (c), nitrogen content (N %), viscosity-average molecular weight (M(η)), temperature (T), and time (t) were analyzed and discussed in detail. The sol-gel transition temperature of CC (M(η) = 7.78 × 10(4)) solution decreased from 36.5 to 31.3 °C with an increase of the concentration from 3.0 to 4.3 wt % and decreased from 35.7 to 27.5 °C with an increase of the nitrogen content from 1.718 to 5.878%. The gelation temperature of a 3.8 wt % CC solution dropped from 38.2 to 34.4 °C with the M(η) of CC increased from 6.35 × 10(4) to 9.56 × 10(4). The gelation time of the CC solution was relatively short at 30 °C, but the solution was stable for a long time at about 15 °C. Moreover, the gels already formed at elevated temperature were irreversible; that is, after cooling to a lower temperature including the dissolution temperature (-7 °C), they could not be dissolved to become liquid.     

Electrically conductive bacterial cellulose by incorporation of carbon nanotubes

Electrically conducting polymeric membranes were prepared by incorporating multiwalled carbon nanotubes (MWCNTs) into bacterial cellulose pellicles produced by Gluconacetobacter xylinum. The MWCNTs were dispersed in a surfactant (cationic cetyl trimethylammonium bromide) solution, and cellulose pellicles were dipped into the solution for 6, 12, and 24 h. The surfactants were then extracted in pure water and dried. Electron microscopy showed that the individual MWCNTs were strongly adhered to the surface and the inside of the cellulose pellicle. The conductivity of the MWCNTs-incorporated cellulose pellicle, as measured by a four-probe at room temperature, was 1.4 x 10(-1) S/cm, based on the total cross-sectional area (approximately 9.6 wt % of MWCNTs). This suggests that the MWCNTs were incorporated uniformly and densely into the pellicles.     

Preparation of ultrafine oxidized cellulose mats via electrospinning

Ultrafine oxidized cellulose (OC) mats were prepared by oxidation of ultrafine cellulose mats produced by electrospinning and subsequent deacetylation of cellulose acetate for potential applications in nonwoven adhesion barriers. When ultrafine cellulose mats were oxidized with a mixture of HNO3/H3PO4 - NaNO2 (2/1/1.4 v/v/wt %), their ultrafine mat structure remained unchanged. The yield and carboxyl content of OC mats were 86.7% and 16.8%, respectively. OC showed lower crystallinity than cellulose because the oxidation of cellulose proceeded via disruption of hydrogen bonds between cellulose chains. The swelling behaviors of ultrafine OC mats were dependent on the type of swelling solution. In a physiological salt solution, their degree of swelling was approximately 230%.     

Effects of modified cellulose nanocrystals on the barrier and migration properties of PLA nano-biocomposites

The aim of this paper is to report the impact of the addition of cellulose nanocrystals on the barrier properties and on the migration behaviour of poly(lactic acid), PLA, based nano-biocomposites prepared by the solvent casting method. Their microstructure, crystallinity, barrier and overall migration properties were investigated. Pristine (CNC) and surfactant-modified cellulose nanocrystals (s-CNC) were used, and the effect of the cellulose modification and content in the nano-biocomposites was investigated. The presence of surfactant on the nanocrystal surface favours the dispersion of CNC in the PLA matrix. Electron microscopy analysis shows the good dispersion of s-CNC in the nanoscale with well-defined single crystals indicating that the surfactant allowed a better interaction between the cellulose structures and the PLA matrix. Reductions of 34% in water permeability were obtained for the cast films containing 1wt.% of s-CNC while good oxygen barrier properties were detected for nano-biocomposites with both 1wt.% and 5wt.% of modified and un-modified cellulose nanocrystals, underlining the improvement provided by cellulose on the PLA films. Moreover, the migration level of the studied nano-biocomposites was below the overall migration limits required by the current normative for food packaging materials in both non-polar and polar simulants.     

Investigation of a 2-step agglomeration process performed in a rotary processor using polyethylene glycol solutions as the primary binder liquid

The purpose of this research was to investigate the use of polyethylene glycol (PEG) solutions as the primary binder liquid in a 2-step agglomeration process performed in a rotary processor and characterize the resulting granules and their tableting characteristics. This was done by granulation of binary mixtures of microcrystalline cellulose (MCC) and either lactose, calcium phosphate, acetaminophen, or theophylline, in a 1∶3 ratio, using a 50% (wt/wt) aqueous solution of PEG and water as the binder liquid. Formulations containing lactose were agglomerated using 5 different amounts of the PEG binder solution, giving rise to a PEG content in the range of 6% to 43% (wt/wt). The process outcome was characterized according to adhesion, yield, and water requirement, and the prepared granules were characterized according to size, size distribution, and flow properties as well as tableting properties. The agglomeration of all mixtures resulted in high yields of free-flowing agglomerates and gave rise to good reproducibility of the investigated agglomerate characteristics. The process allowed for the incorporation of 42.5% (wt/wt) PEG, which is higher than the percentage of PEG reported for other equipment. Tables of sufficient strength could be prepared with all investigated excipients using 20% wt/wt PEG; higher PEG contents gave rise to adhesion and prolonged disintegration. In conclusion, agglomeration in a torque-controlled rotary processor using solutions of PEG as the primary binder liquid was found to be a robust process, suitable for the incorporation of high contents of PEG and/or drug compounds.     

Simulation and operation performance of simulated moving bed for enatioseparation of mandelic acid

Separation of mandelic acid enantiomer was carried out by using laboratory simulated moving bed (SMB) chromatography. The SMB process consists of four zones, with each zone having 1 column. The triangle theory was used to obtain the operating conditions for SMB. Adsorption isotherms of the D-/L-mandelic acids were obtained by pulse input method, considering linear isotherms of the two components. Flow rates of extract, raffinate, feed, and eluent streams were systematically changed to understand the effects of operation flow rates of SMB. Simulation results and experimental data from the SMB chromatography showed good agreements. Adjusting the flow rate in zone II increased the purity of D-mandelic acid from extract port. The highest purity of D-mandelic acid in the extract was obtained as 94% under the operating flow rates of Q_feed = 0.1 mL/min, Q_extract = 0.2 mL/min, Q_raffinate = 0.8 mL/min, and Q_eluent = 0.9 mL/min.     

Studies on chitin. 2. Preparation and properties of chitin membranes

A chitin membrane was prepared by a new procedure involving coagulation of the chitin solution in N,N-dimethyl acetamide, N-methyl 2-pyrrolidone and lithium chloride (DMA-NMP-LiCl) with 2-propanol. The solute permeability, water sorption and mechanical properties were compared with membranes prepared by two previously reported methods (coagulation of a formic acid and dichloroacetic acid (FA-DCA) solution of chitin with 2-propanol; and coagulation of a trichloroacetic acid and dichloroethane (TCA-DCE) solution of chitin with acetone). The permeability coefficients of the three chitin membranes were higher than a regenerated cellulose membrane (Cuprophane®). The membrane prepared from DMA-NMP-LiCl solution had a higher tensile strength (3·3 Mpa) in the wet state than the others. The membrane obtained from TCA-DCE solution absorbed more water (360%) and the membrane prepared from FA-DCA solution was relatively weak (1·8 MPa) in the wet state. It was suggested that 2-propanol was a favourable coagulant for membrane production. In addition, the effect of the origin of chitin on molecular weight and tensile properties of the membranes was studied.     

Characterization of Chinese hamster ovary cell culture feed media precipitate

Process intensification of monoclonal antibody production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution, changing the media composition and leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the precipitated components so that mitigation strategies can be developed. From the conducted analysis, the precipitate was predominately found to be organic and was analyzed with liquid chromatography-mass spectrometry and inductively coupled plasma-optical emission spectroscopy (ICP-OES) to identify the constituent components. Up to ten amino acids were identified with tyrosine (approximately 77 wt.%) and phenylalanine (approximately 4 wt.%) being the most prevalent amino acids. Elemental analysis with ICP-OES revealed that inorganic components were accounted for less than one weight percentage of the solid precipitate with metal sulfates being the predominant inorganic components.     

Clostridium lentocellum SG6--a potential organism for fermentation of cellulose to acetic acid

A cellulolytic, acetic acid producing anaerobic bacterial isolate, Gram negative, rod-shaped, motile, terminal oval shaped endospore forming bacterium identified as Clostridium lentocellum SG6 based on physiological and biochemical characteristics. It produced acetic acid as a major end product from cellulose fermentation at 37°C and pH 7.2. Acetic acid production was 0.67 g/g cellulose substrate utilized in cellulose mineral salt (CMS) medium. Yeast extract (0.4%) was the best nitrogen source among the various nitrogenous nutrients tested in production medium containing 0.8% cellulose as substrate. No additional vitamins or trace elemental solution were required for acetic acid fermentation. This is the highest acetic acid fermentation yield in monoculture fermentation for direct conversion of cellulose to acetic acid.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Rheological properties and molecular structure of tunicate cellulose in LiCl/1,3-dimethyl-2-imidazolidinone

Solution properties and molecular structure of tunicate cellulose (TC), an animal cellulose from Halocynthia roretzi, were investigated in terms of rheological and dilute solution properties. The solvent used is 8 wt % LiCl/1,3-dimethyl-2-imidazolidinone (DMI). A solution of dissolving pulp (DP), derived from plant, was also used for comparison. The weight-average molecular weight, Mw, and the limiting viscosity number, [eta], of the TC were evaluated to be 413 x 10(6) and 2645 mL/g, respectively. The TC solution showed the same concentration dependence of GN (GN=5.49 x 10(6)phiw(2.1)4 Pa; phiw: weight fraction of cellulose in solution; GN: plateau modulus) as the DP solution and, moreover, also as the solution of cotton linter (CC) in 8 wt % LiCl/N,N-dimethylacetamide (DMAc). This exponent of 2.1(4) indicates that network structure by entanglements was formed in these solutions. According to the theory of Fetters et al., moreover, such identity means that all of these celluloses have the identical chain structure though their biological origins are far different. On the other hand, the phiw-dependence of eta0-etas (eta0=zero shear rate viscosity of solution; etas=solvent viscosity) was different between the TC and the DP solution in the semidilute regime: the TC solution exhibited eta0-etas proportional, variant phiw(7.5) and the DP solution eta0-etas proportional, variant phiw4. According to the theory of Doi-Edwards, this exponent of 4 (the DP solution) indicates that the DP behaves as flexible polymers in the solution. In contrast, the dependence for the TC solution seems unexplainable on the basis of molecular theories. This difference probably signifies the difference in the relaxation process or mechanism in entanglement systems.