Graft copolymerization of methyl acrylate onto pullulan using ceric ammonium nitrate as initiator

Water absorption resins of pullulan graft methyl acrylate (PU-g-MA) using ceric ammonium nitrate (CAN) as an initiator has been investigated under nitrogen atmosphere in aqueous medium. The percentage grafting (%PG) is favoured by increasing reaction time but is affected by higher concentration of initiator and monomer, and high temperature. Experiments showed that the optimal conditions for grafting were: [CAN] = 0.004 mol/l which was added in 1 mol/l nitric acid; [MA] = 0.0465 mol/l; reaction time; 180 min and temperature, 40 °C. The graft copolymer was analyzed by infrared spectroscopy. The water absorption capacity of the resins decreased significantly with the increase in %PG.     

Synthesis of poly-(ε)-caprolactone grafted starch co-polymers by ring-opening polymerisation using silylated starch precursors

Poly-(ε)-caprolactone grafted corn starch co-polymers were synthesized using a hydrophobised silylated starch precursor. The silylation reaction was performed using hexamethyl disilazane (HMDS) as the reagent in DMSO at 70 °C. Silylated starch with a degree of substitution (DS) between 0.45 and 0.7 was obtained. ε-Caprolactone is grafted to silylated starch by a ring-opening polymerisation catalysed by Al(OiPr)₃ in THF at 50 °C. The grafting efficiency varies between 28% and 58%, the remainder being homopolymers of ε-caprolactone. The DS of the polycaprolactone graft is between 0.21 and 0.72. The poly-(ε)-caprolactone side chains consist of 40–55 monomer units and is a function of the reagent intakes. Experiments with native starch under similar conditions do not result in the desired poly-(ε)-caprolactone grafted corn starch co-polymers and unreacted starch was recovered after work-up. Removal of the silyl groups of the poly-(ε)-caprolactone grafted starch co-polymers is possible using a mild acid treatment with diluted hydrochloric acid in THF at room temperature.     

Xanthan-g-poly(acrylamide): Microwave-assisted synthesis,characterization and in vitro release behavior

Xanthan-g-poly(acrylamide) was synthesized employing microwave-assisted and ceric-induced graft copolymerization, and was characterized by FT-IR, DSC, XRD and SEM studies. Matrix tablets of diclofenac sodium were formulated using graft copolymer as the matrix by direct compression technique. Release behavior of the graft copolymer was evaluated using USP type-II dissolution apparatus in 900 ml of phosphate buffer (pH 6.8), maintained at 37 °C and at 50 rpm. Microwave-assisted grafting provided graft copolymer with higher % grafting in a shorter time in comparison to the ceric-induced grafting. The % grafting was found to increase with the increase in the power of microwave and/or time of exposure. The matrix tablets were found to release the drug by zero-order kinetics, and the faster release of drug was observed from the graft copolymer matrix as compared to the xanthan gum matrix. It was observed that grafting reduces the swelling, but increases the erosion of xanthan gum.     

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.     

Immobilization of urease using glycidyl methacrylate grafted nylon-6-membranes

The graft copolymerization of glycidyl methacrylate (GMA) onto nylon-6-membrane using benzophenone (BP) as an initiator was carried out in an alcoholic aqueous solution. The acrylic double bond of GMA participated in the grafting onto the nylon-6-membrane backbone with the epoxy groups remaining unaffected. At the end of the grafting reaction, urease was immobilized onto the modified membrane. BP concentration, GMA concentration and organic solvent seperation were studied by determining the grafting percentage. The influence of urease concentration on the immobilization efficiency was also studied. With keeping other conditions constant, the optimum conditions were shown as following [BP]: 5 × 10⁻² mM; [GMA]: 10 M; [urease]: 10 mg/ml, organic solvent: methanol.     

Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria

An extracellular protease from a newly isolated seawater haloalkaliphilic bacterium, haloalkaliphilic bacteria Ve₂-20-9₁ [HM047794], was purified and characterized. The enzyme is a monomer with a 37.2 kDa estimated molecular weight. It catalyzed reactions in the pH range 8–11 and performed optimally at pH 10. While maximal activity occurred at 50 °C, the temperature profile shifted from 50 to 80 °C in 1–3 M NaCl. The enzyme's thermal stability was probed using circular dichroism (CD) spectroscopy with NaCl at 50 and 70 °C. The changes in the enzyme's secondary structure were also analyzed using Fourier transform infrared spectroscopy (FTIR). The N-terminal amino acid sequence GKDGPPGLCGFFGCI exhibited low homology with other bacterial proteases, which highlights the enzyme's novelty. The enzyme was labile in anionic surfactant (1% w/v SDS) but showed stability in non-ionic surfactants (Tween 20, Tween 80 and Triton X-100 all 1% v/v), commercial detergents, and oxidizing and reducing agents. The enzyme's excellent stability in commercial detergents highlights its potential as a detergent additive.     

Effects of triterpene derivatives from Maytenus rigida on VEGF-induced Kaposi's sarcoma cell proliferation

Betulinic acid (BA) is a naturally occurring lupane-type triterpene which exhibits a variety of biological activities including potent cytotoxic properties. On the basis of the structural similarity to BA, two lupane derivatives namely lup-20(29)-ene-3β,30-diol (1) and lup-20(29)-ene-3β,28-diol (2), along with two friedelane derivatives, namely friedelan-3-one (3) and friedelan-3β-ol (4), isolated from the Brazilian plant Maytenus rigida, have been evaluated for their anti-proliferative effect. Similarly to BA, compounds 1 and 3 at 1 μM concentration significantly inhibited the VEGF-induced Kaposi's sarcoma (KS) cell proliferation by 50%. In contrast, this effect was not found in control endothelial cells (EC).Moreover, compounds 1 and 3 showed a dose-dependent effect on the apoptotic cell death, as detected by FACS analysis and caspase-3 assay. Specifically, at 10 μM concentration, apoptosis was significantly induced (from 45% to 55% of hypodiploid cells vs control cells) and showed the same potency order observed for the anti-proliferative effect at 1 μM, i.e., compound 3 > BA > compound 1.Taking into account the interest given rise by BA as anticancer agent, the comparable anti-proliferative activity shown by compounds 1 and 3 and BA, can give an impulse to further investigate lupane and friedelane derivatives as cytotoxic agents.     

Debranching enzyme concentration effected on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from amylose rice starch

The effect of debranching enzyme concentration on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from high amylose rice starch were studied. The pullulanase enzyme (8, 10, 12, 14 and 16 U/g starch) was introduced to modify amylopectin molecules of 15% (w/w) gelatinized rice starches at 55 °C for 16 h. The debranched starches with different degrees of hydrolysis (0.14–5.27%), and having 66.60–98.82% β-amylolysis limit were then induced at 4 °C for 16 h, afterward a one cycle of freeze–thaw process (?10/30 °C) was applied. The results showed that a pullulanase hydrolysis improved the degree of syneresis (51.64–54.85% from 8 to 16 U/g starch). Resistant starch content increased sharply as the amount of the enzyme increased, reaching the highest (19.81%) for a 12 U/g starch and decreased to 13.16% by 16 U/g starch. α-Amylase hydrolysis rate showed that incompletely-debranched had a lower estimated glycemic index than completely debranched rice starches. Microstructure of the selected RS III samples using X-ray diffraction and scanning electron microscopy revealed a crystal pattern change from A- to V-type pattern and formed a coarse honeycomb-like and a filamentous network structure.     

Free radical copolymerization kinetics of γ-methyl-α-methylene-γ-butyrolactone (MeMBL)

The propagation kinetics and copolymerization behavior of the biorenewable monomer γ-methyl-α-methylene-γ-butyrolactone (MeMBL) are studied using the pulsed laser polymerization (PLP)/size exclusion chromatography (SEC) technique. The propagation rate coefficient for MeMBL is 15% higher than that of its structural analogue, methyl methacrylate (MMA), with a similar activation energy of 21.8 kJ·mol(-1). When compared to MMA, MeMBL is preferentially incorporated into copolymers when reacted with styrene (ST), MMA, and n-butyl acrylate (BA); the monomer reactivity ratios fit from bulk MeMBL/ST, MeMBL/MMA, and MeMBL/BA copolymerizations are r(MeMBL) = 0.80 ± 0.04 and r(ST) = 0.34 ± 0.04, r(MeMBL) = 3.0 ± 0.3 and r(MMA) = 0.33 ± 0.01, and r(MeMBL) = 7.0 ± 2.0 and r(BA) = 0.16 ± 0.03, respectively. In all cases, no significant variation with temperature was found between 50 and 90 °C. The implicit penultimate unit effect (IPUE) model was found to adequately fit the composition-averaged copolymerization propagation rate coefficient, k(p,cop), for the three systems.     

Graft polymers of eucalyptus lignosulfonate calcium with acrylic acid: Synthesis and characterization

The graft copolymerization of eucalyptus lignosulfonate calcium (HLS-Ca) from hardwood and acrylic acid (AA) was investigated by using Fenton agent as a coinitiator. The influences of reaction conditions on grafting parameters i.e. product yield (Y%), AA conversion (C%), grafting ratio (G%) and grafting efficiency (GE%) were carefully studied. The effects of the phenolic hydroxyl (Ph-OH) group on the polymerization of AA and grafting reaction were researched. Graft copolymers were identified by the new absorption at 1727 cm^?1, more homogenized morphology and higher decomposition temperature after grafted with AA, as illustrated in FTIR, SEM and TG spectra. The optimum synthesis conditions are as follows: H₂O₂ = 25.2 mol/L, FeCl₂ = 63.0 mol/L, T = 50 °C and t = 2 h and the optimum percentages of Y, C, G and GE are 97.61%, 95.23%, 71.29% and 78.85%, respectively. The Ph-OH group of HLS-Ca cannot inhibit the polymerization of AA and is involved in the grafting reaction as an active center.     

Digestibility,nitrogen balance,and blood metabolites in llama (Lama glama) and alpaca (lama pacos) fed barley or barley alfalfa diets

To determine the effect of barley diets on digestibility, nitrogen balance, and blood metabolites, mature gelded llamas and alpacas (n = 8; 4 llamas, 36 ± 4 months, 90 ± 10.7 kg; 4 alpacas, 24–36 months, 50 ± 4 kg) were randomly fed 100% barley (B) and 20% alfalfa/80% barley (BA) hay. Animals were housed in metabolism crates and diets were fed for a 7 days adjustment period followed by a 5 days collection period. Feed, feed refusal, feces and urine were collected, dried and N content determined by combustion analysis. Blood samples were collected on day 12 at 30 min intervals over a 6 h period. Plasma was harvested and analyzed for electrolytes (Na, K, Cl, Ca, Ca²⁺, P, Mg), metabolites glucose, non-esterified fatty acids (NEFAs), urea N, creatinine, albumin, total protein (TPP), osmolality (Osm). Plasma glucose, urea N, albumin, osmolality, electrolyte and metabolite levels were similar between species, and were unaffected by diet. On a metabolic weight basis, only diet was significant for N intake, urinary and fecal N, and total N excreted. Dry matter intake was not significantly different; however, BA consumption was greater than B, (B) 1272 g N/day and (BA) 1636 g N/day for llamas, and for alpacas (B) 835 g N/day and (BA) 1034 g N/day, respectively. Nitrogen intake followed the same pattern, (B) 21.4 g N/day and (BA) 33.9 g N/day, respectively for llamas, and (B) 13.6 g N/day and (BA) 20.6 g N/day, respectively for alpacas (diet, P < 0.002). Diet affects were significant for urine N excretion (P < 0.02), (B) 11.2 g/day and (BA) 18.2 g/day for llamas, and (B) 6.8 and (BA) 10.8 g N/day for alpacas. Fecal N excretion was different for diet (P < 0.03), with fecal excreted N of 9.0 g N/day and 11.9 g N/day for B and BA in llamas, and 5.9 g N/day and 9.1 g N/day for B and BA respectively in for alpacas, respectively. Nitrogen retention, DM digestibility and N digestibility were unaffected by diet or species. However, the llamas in this study displayed an increase in nitrogen intake of 64.6% between the B and BA diets with a 381% increase in N retention. Alpacas increased their N intake by 57.4% when they consumed the BA forage, which only increased N retention by 22.2%. These species differences indicate that alpacas have a lower N requirement to meet metabolic needs than llamas, which are likely related to the smaller body size of the alpaca. When examining the biological value of N from the respective diets, alpacas and llamas had a value of 56.2% when consuming barley. The BA diet had a higher biological value of 65.0% in llamas compared to 57.4% in alpacas. Therefore, on the basis of this study, extrapolations between llamas and alpacas with respect to nitrogen requirement and balance are not valid.     

Cloning,expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes, belong to glycoside hydrolase family 13. A gene corresponding to MA in Geobacillus caldoxylosilyticus TK4 (GcaTK4MA) was cloned into pET28a(+) vector and expressed in Escherichia coli with 6xHis-tag at the N-terminus. Herein, we report on the biochemical properties of a new thermo- and pH-stable MA. GcaTK4MA has similar properties those of other MAases in terms of the primary structure, preference for CD over starch and having an extra domain at its N- and C-terminals. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. The purified enzyme exhibited optimal activity for β-CD hydrolysis at 50 °C and pH 7.0. When the enzyme was separately incubated at 4 °C and 50 °C in the buffer solutions (pH 3.0–9.0) up to 7 days, it was seen that the enzyme had the higher stability at 50 °C than 4 °C. The enzyme retained about 80% of its original activity when it was incubated at 50 °C for 7 days. The enzyme activity was significantly inhibited by SDS and EDTA at the final concentration of 1%. These results suggest that this is the first reported MA having an extremely pH- and thermal stabilities.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Biodegradability and mechanical properties of polycaprolactone composites encapsulating phosphate-solubilizing bacterium Bacillus sp. PG01

This study examined the feasibility of using polycaprolactone (PCL) and its composites (with starch and/or clay) in encapsulating cells of phosphate-solubilizing bacteria (PSB) for the development of biodegradable and “controlled-release” bacterial fertilizer. The PSB used in this work was an indigenous Bacillus sp. PG01 isolate. The results show that the PG01 strain was able to degrade all the cell-loaded capsules made of PCL and PCL composites, resulting in a continual cell release. Morphology observation indicates that severe disruption of the capsule structure occurred after incubation for 15–20 days. The biodegradability of the capsules decreased in the order of PCL/starch (20 wt%) > PCL/starch (20 wt%)/cay (7 wt%) > PCL alone > PCL/clay (7 wt%). Similar trends were also observed for the decrease in tensile strength and elongation at break, suggesting strong connections between biodegradability and the mechanical properties. Addition of starch appeared to enhance the biodegradability of the capsules, whereas the clay-blended composites were less biodegradable. The amount and rate of cell release from cell-encapsulated PCL-based capsules were positively dependent on the biodegradability and on the decrease in the mechanical strength. Nevertheless, the pattern of cell release was quite similar for all types of capsules. The outcome of this work seems to suggest that by proper manipulation of composite compositions, the controlled release of the bacterial fertilizer (i.e., Bacillus sp. PG01 cells) might be achievable.     

Expression of fibroblast growth factor 21 in patients with biliary atresia

Fibroblast growth factor 21 is a critical circulating adipokine involving in metabolic disorders and various liver diseases. This study was performed to investigate whether FGF21 is also associated with the pathophysiology of biliary atresia. Serum FGF21 levels were measured in 57 BA patients and 20 age matched healthy controls. We also examined hepatic FGF21 mRNA expression and FGF21 protein levels in liver tissues obtained from 15 BA patients undergoing liver transplantation and 5 cases of pediatric donation after cardiac death donor without liver diseases by RT-PCR and Western blotting. Patients with BA showed significantly higher serum FGF21 levels than those without BA (554.7 pg/mL [83–2300] vs. 124.5 pg/mL [66–270], P < 0.05). Patients with BA also had significantly higher FGF21 mRNA and protein levels in hepatic tissues than control subjects. Serum FGF21 expression increased corresponding to the severity of liver fibrosis. Furthermore, serum FGF21 levels dropped significantly in BA patients within 6 months after liver transplantation and approached baseline in healthy controls (P > 0.05). In vivo, FXR knockout could significantly abrogate cholestasis induced FGF21 expression. FGF21 levels in serum and liver tissue increased significantly in BA patients. In vivo, cholestasis could induce FGF21 expression in FXR dependent manner.     

Changes in starch structure during manufacturing of starch microspheres for use in parenteral drug formulations: Effects of temperature treatment

Starch microspheres were produced by emulsification of a starch dispersion in an aqueous polyethylene glycol (PEG) solution. Crystalline/ordered structure was formed within these starch droplets during incubation at 6 °C for 25 h followed by incubation at 37 °C for 28 h. After incubation at 37 °C the crystalline structure in the samples was of type B. The crystallization process of microspheres was compared with crystallization in a model system. The crystalline structure of the microspheres melted at temperatures almost 20 °C lower than in the model system incubated under the same conditions, as determined by differential scanning calorimetry. It was thus concluded that the crystallization process within microspheres was different than that of bulk starch and the ability of the starch molecules to reorganize themselves within the dispersed starch phase of an aqueous two-phase system at the higher incubation temperature was limited. It was also observed that the presence of PEG or carbonate buffer protected the molecular order formed by the starch molecules during incubation from breakdown during freeze-drying.     

Starch pastes thinning during high-pressure homogenization

High-pressure homogenization induced thinning of potato and cassava starch paste was investigated. The starch slurries at a concentration of 2.0 wt.% were heated at 90 °C for 1 h and then rapidly cooled in tap water. The cooled starch pastes were homogenized at various pressures ranging from 0 to 100 MPa using a lab-scale high-pressure homogenizer. The influence of homogenizing pressure on the temperature, apparent viscosity, electrical conductivity, and percent light transmittance of homogenized starch pastes were determined. Temperatures of homogenized starch pastes increased linearly with the increase of the applied pressure, and the rate was 0.177 °C/MPa and 0.186 °C/MPa for potato and cassava starch pastes, respectively. After high-pressure homogenization, the apparent viscosities of the starch pastes decreased, while the percent light transmittances of them increased. However, the electrical conductivities of starch pastes were not affected by homogenization.     

Microwave-assisted modification of starch for compatibilizing LLDPE/starch blends

Modification of activated cassava starch (S) was performed by using octenyl succinic anhydride (OSA) at different starch/OSA ratios under microwave radiation. FTIR and titration results indicated that, within a reaction time of 7 min, degrees of substitution (DS) of about 0.045 may be achieved with 20% OSA. Subsequently, linear low density polyethylene/starch (LLDPE/S) blends were prepared employing succinylated starches (S-g-OSA) as compatibilizers. The morphology and mechanical properties of LLDPE/S blends with and without compatibilizer were compared. It was observed that the addition of 10% of compatibilizer with respect to the dispersed phase content led to a reduction of the starch phase size and to an improvement of the blends mechanical properties.     

Some properties of white and yellow plantain (Musa paradisiaca,Normalis) starches

Starch obtained from yellow and white plantain varieties were subjected to proximate analysis, physicochemical and rheological characterization in order to evaluate their properties. Yellow plantain variety gave higher yield of starch than the white variety. The two varieties differed in the purity of starch extract; white plantain starch contained: ash (1.09%), protein (0.640%) and fat (0.276%) while yellow plantain starch contained: ash (0.95%), protein (0.325%) and fat (0.403%). The amylose content of yellow plantain starch (24.36% (apparent), 26.13% (total)) was similar to that of white plantain starch (24.24% (apparent), 26.01% (total)). Scanning electron microscopy revealed bimodal irregular shaped granules (3.74–7.00 and 10.00–33.00 μm) in white plantain starch and elliptical granules (11.22–41.00 μm) in yellow plantain starch. Both starches differed markedly in their physicochemical properties. Their differences in gelatinization temperature (yellow plantain, 64.99–73.90 °C; white plantain, 68.08–77.15 °C), swelling and solubility patterns, and pasting characteristics indicated that yellow plantain starch had weaker granule architecture compared with white plantain starch. Further evidence of differences in properties was obtained from flow and viscoelastic properties of the starch gels, paste clarity and freeze–thaw stability.     

Production of acetone–butanol–ethanol (ABE) in a continuous flow bioreactor using degermed corn and Clostridium beijerinckii

An examination of the sustainability of the long-term cultivation of C. beijerinckii BA101 in degermed corn/saccharified degermed corn based P2 medium has been described in this work. It was found that long-term continuous cultivation of C. beijerinckii BA101 in a degermed corn based medium was not possible due to the instability of the gelatinized degermed corn starch during storage often called “retrogradation”. Using this substrate, continuous ABE fermentation was run for 228 h, before the fermentation turned acidogenic. However continuous fermentations of saccharified degermed corn with normal and half P2 medium nutrients were successful. In saccharified degermed corn continuous fermentation, ABE concentration up to 14.28 g/L was achieved at a dilution rate of 0.03 h⁻¹. This work demonstrated that byproduct (germ/oil, corn fiber) credit can be obtained by fermenting saccharified degermed corn in continuous flow bioreactors. Additionally significant savings can be achieved by supplementing with half of normal P2 medium nutrients.