Search results for: glutaraldehyde

Authors: Sperisa Distantina, Fadilah Fadilah, Mujtahid Kaavessina

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Hydrogel films were prepared from kappa carrageenan by crosslinking with glutaraldehyde. Carrageenan films extracted from Kappaphycus alvarezii seaweed were immersed in glutaraldehyde solution for 2 min and then cured at 110 °C for 25 min. The obtained crosslinked films were washed with ethanol to remove the unreacted glutaraldehyde and then air dried to constant weights. The aim of this research was to study the swelling degree behaviour of the hydrogel film to neutral salts solution, namely NaCl, KCl, and CaCl₂. The results showed that swelling degree of crosslinked films varied non-monotonically with salinity of NaCl. Swelling degree decreased with the increasing of KCl concentration. Swelling degree of crosslinked film in CaCl₂ solution was lower than that in NaCl and in KCl solutions.

Keywords: carrageenan, hydrogel, glutaraldehyde, salt, swelling

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Authors: Sperisa Distantina, Fadilah, Mujtahid Kaavessina

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Crosslinked konjac glucomannan and kappa carrageenan film were prepared by chemical crosslinking using glutaraldehyde (GA) as the crosslinking agent. The effect crosslinking on the swelling degree was investigated. Konjac glucomanan and its mixture with kappa carragenan film was immersed in GA solution and then thermally cured. The obtained crosslinked film was washed and soaked in the ethanol to remove the unreacted GA. The obtained film was air dried at room temperature to a constant weight. The infrared spectra and the value of swelling degree of obtained crosslinked film showed that glucomannan and kappa carrageenan was able to be crosslinked using glutaraldehyde by film immersion and curing method without catalyst. The crosslinked films were found to be pH sensitive, indicating a potential to be used in drug delivery polymer system.

Keywords: crosslinking, glucomannan, carrageenan, swelling

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Authors: Sperisa Distantina, Rieke Ulfha Noviyanti, Sri Sutriyani, Fadilah Fadilah, Mujtahid Kaavessina

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In this research, carrageenan extracted from seaweed Eucheuma cottonii was mixed with polyvinyl alcohol (PVA) and then crosslinked using glutaraldehyde (GA). The obtained hydrogel films were applied to control the drug release rate of paracetamol. The aim of this research was to develop a mathematical model that can be used to describe the mass transfer rate of paracetamol from the hydrogel film into buffer solution. The effect of weight ratio carrageenan-PVA (5: 0, 1: 0.5, 1: 1, 1: 2, 0: 5) on the parameters of the mathematical model was investigated also. Based on the experimental data, the proposed mathematical model could describe the mass transfer rate of paracetamol. The weight ratio of carrageenan-PVA greatly affected the amount of paracetamol absorbed in the hydrogel film and the mass transfer rate of paracetamol.

Keywords: carrageenan-PVA, crosslinking, glutaraldehyde, hydrogel, paracetamol, mass transfer

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Authors: Bipasa Dey, Varsha Panwar, Tanmay Dutta

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Laccase, a multicopper oxidase (EC 1.10.3.2) have been at the forefront as a superior industrial biocatalyst. They are versatile in terms of bestowing sustainable and ecological catalytic reactions such as polymerisation, xenobiotic degradation and bioremediation of phenolic and non-phenolic compounds. Regardless of the wide biotechnological applications, the critical limiting factors viz. reusability, retrieval, and storage stability still prevail. This can cause an impediment in their applicability. Crosslinked enzyme aggregates (CLEAs) have emerged as a promising technique that rehabilitates these essential facets, albeit at the expense of their enzymatic activity. The carrier free crosslinking method prevails over the carrier-bound immobilisation in conferring high productivity, low production cost owing to the absence of additional carrier and circumvent any non-catalytic ballast which could dilute the volumetric activity. To the best of our knowledge, the ε-amino group of lysyl residue is speculated as the best choice for forming Schiff’s base with glutaraldehyde. Despite being most preferrable, excess glutaraldehyde can bring about disproportionate and undesirable crosslinking within the catalytic site and hence could deliver undesirable catalytic losses. Moreover, the surface distribution of lysine residues in Trametes versicolor laccase is significantly less. Thus, to mitigate the adverse effect of glutaraldehyde in conjunction with scaling down the degradation or catalytic loss of the enzyme, crosslinking with inert substances like gelatine, collagen, Bovine serum albumin (BSA) or excess lysine is practiced. Analogous to these molecules, sugars have been well known as a protein stabiliser. It helps to retain the structural integrity, specifically secondary structure of the protein during aggregation by changing the solvent properties. They are comprehended to avert protein denaturation or enzyme deactivation during precipitation. We prepared crosslinked enzyme aggregates (CLEAs) of laccase from T. versicolor with the aid of sugars. The sugar CLEAs were compared with the classic BSA and glutaraldehyde laccase CLEAs concerning physico-chemical properties. The activity recovery for the fructose CLEAs were found to be ~20% higher than the non-sugar CLEA. Moreover, the 𝐾𝑐𝑎𝑡𝐾𝑚⁄ values of the CLEAs were two and three-fold higher than BSA-CLEA and GACLEA, respectively. The half-life (t1/2) deciphered by sugar-CLEA was higher than the t1/2 of GA-CLEAs and free enzyme, portraying more thermal stability. Besides, it demonstrated extraordinarily high pH stability, which was analogous to BSA-CLEA. The promising attributes of increased storage stability and recyclability (>80%) gives more edge to the sugar-CLEAs over conventional CLEAs of their corresponding free enzyme. Thus, sugar-CLEA prevails in furnishing the rudimentary properties required for a biocatalyst and holds many prospects.

Keywords: cross-linked enzyme aggregates, laccase immobilization, enzyme reusability, enzyme stability

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Authors: Jen-Taut Yeh, Yuan-jing Hou, Li Cheng, Ya Zhou Wang, Zhi Yu Zhang

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Tensile retention properties of bacterial cellulose (BC) reinforced thermoplastic starch (TPS) resins were successfully improved by reacting with glutaraldehyde (GA) in their gelatinization processes. Small amounts of poly (lactic acid) (PLA) were blended with GA modified TPS resins to improve their processability. As evidenced by the newly developed ether (-C-O-C-) stretching bands on FT-IR spectra of TPS100BC0.02GAx series specimens, hydroxyl groups of TPS100BC0.02 resins were successfully reacted with the aldehyde groups of GA molecules during their modification processes. The retention values of tensile strengths (σf) of TPS100BC0.02GAx and (TPS100BC0.02GAx)75PLA25 specimens improved significantly and reached a maximal value as GA contents approached an optimal value at 0.5 part per hundred parts of TPS resin (PHR). By addition of 0.5 PHR GA in biocomposite specimens, the initial tensile strength and elongation at break values of (TPS100BC0.02GA0.5)75PLA25 specimen improved to 24.6 MPa and 5.6%, respectively, which were slightly improved than those of (TPS100BC0.02)75PLA25 specimen. However, the retention values of tensile strengths of (TPS100BC0.02GA0.5)75PLA25 specimen reached around 82.5%, after placing the specimen under 20oC/50% relative humidity for 56 days, which were significantly better than those of the (TPS100BC0.02)75PLA25 specimen. In order to understand these interesting tensile retention properties found for (TPS100BC0.02GAx)75PLA25 specimens. Thermal analyses of initial and aged TPS100BC0.02, TPS100BC0.02GAx and (TPS100BC0.02GAx)75PLA25 specimens were also performed in this investigation. Possible reasons accounting for the significantly improved tensile retention properties of TPS100BC0.02GAx and (TPS100BC0.02GAx)75PLA25 specimens are proposed.

Keywords: biocomposite, strength retention, thermoplastic starch, tensile retention

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Authors: S. Korkut, M. S. Kilic, E. Erhan

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In order to detect and quantify the phenolic contents of a wastewater with biosensors, two working electrodes based on modified Poly (Pyrrole) films were fabricated. Enzyme horseradish peroxidase was used as biomolecule of the prepared electrodes. Various phenolics were tested at the biosensor. Phenol detection was realized by electrochemical reduction of quinones produced by enzymatic activity. Analytical parameters were calculated and the results were compared with each other.

Keywords: carbon nanotube, phenol biosensor, polypyrrole, poly (glutaraldehyde)

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Authors: Heri Hermansyah, Annisa Kurnia, A. Vania Anisya, Adi Surjosatyo, Yopi Sunarya, Rita Arbianti, Tania Surya Utami

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Lipase is one of biocatalyst which is applied commercially for the process in industries, such as bioenergy, food, and pharmaceutical industry. Nowadays, biocatalysts are preferred in industries because they work in mild condition, high specificity, and reduce energy consumption (high pressure and temperature). But, the usage of lipase for industry scale is limited by economic reason due to the high price of lipase and difficulty of the separation system. Immobilization of lipase is one of the solutions to maintain the activity of lipase and reduce separation system in the process. Therefore, we conduct a study about lipase immobilization with the adsorption-cross linking method using glutaraldehyde because this method produces high enzyme loading and stability. Lipase is immobilized on different kind of resin with the various functional group. Highest enzyme loading (76.69%) was achieved by lipase immobilized on anion macroporous which have anion functional group (OH^‑). However, highest activity (24,69 U/g support) through olive oil emulsion method was achieved by lipase immobilized on anion macroporous-chitosan which have amino (NH₂) and anion (OH^-) functional group. In addition, it also success to produce biodiesel until reach yield 50,6% through interesterification reaction and after 4 cycles stable 63.9% relative with initial yield. While for Aspergillus, niger lipase immobilized on anion macroporous-kitosan have unit activity 22,84 U/g resin and yield biodiesel higher than commercial lipase (69,1%) and after 4 cycles stable reach 70.6% relative from initial yield. This shows that optimum functional group on support for immobilization with adsorption-cross linking is the support that contains amino (NH₂) and anion (OH^-) functional group because they can react with glutaraldehyde and binding with enzyme prevent desorption of lipase from support through binding lipase with a functional group on support.

Keywords: adsorption-cross linking, immobilization, lipase, resin

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Authors: Mahmoud H. Aboul-Ela, Riham R. Mohamed, Magdy W. Sabaa

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Synthesis of cross-linked hydrogels composed of trimethyl chitosan (TMC) and poly(vinyl alcohol) (PVA) in different weight ratios in presence of glutaraldehyde as cross-linking agent. Characterization of the prepared hydrogels was done using FTIR, XRD, SEM and TGA. The prepared hydrogels were investigated as adsorbent materials for some transition metal ions from their aqueous solutions. Moreover, the swell ability of the prepared hydrogels was also investigated in both acidic and alkaline pHs, as well as in simulated body fluid (SBF).

Keywords: trimethyl chitosan, hydrogels, metal uptake, superabsorbent materials

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Authors: A. Dashti, M. Eskandari, L. Farahmand, P. Parvin, A. Jafargholi

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Breast Cancer is one of the most considerable diseases in the United States and other countries and is the second leading cause of death in women. Common breast cancer treatments would lead to adverse side effects such as loss of hair, nausea, and weakness. These complications arise because these cancer treatments damage some healthy cells while eliminating the cancer cells. In an effort to address these complications, laser radiation was utilized and tested as a targeted cancer treatment for breast cancer. In this regard, tissue engineering approaches are being employed by using an electrospun scaffold in order to facilitate the growth of breast cancer cells. Polycaprolacton (PCL) was used as a material for scaffold fabricating because of its biocompatibility, biodegradability, and supporting cell growth. The specific breast cancer cells have the ability to create a three-dimensional cell cluster due to the spontaneous accumulation of cells in the porosity of the scaffold under some specific conditions. Therefore, we are looking for a higher density of porosity and larger pore size. Fibers showed uniform diameter distribution and final scaffold had optimum characteristics with approximately 40% porosity. The images were taken by SEM and the density and the size of the porosity were determined with the Image. After scaffold preparation, it has cross-linked by glutaraldehyde. Then, it has been washed with glycine and phosphate buffer saline (PBS), in order to neutralize the residual glutaraldehyde. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) results have represented approximately 91.13% viability of the scaffolds for cancer cells. In order to create a cluster, Michigan Cancer Foundation-7 (MCF-7, breast cancer cell line) and Michigan Cancer Foundation-10A (MCF-10A, human mammary epithelial cell line) cells were cultured on the scaffold in 24 well plate for five days. Then, we have exposed the cluster to the laser diode 808 nm radiation to investigate the effect of laser on the tumor with different power and time. Under the same conditions, cancer cells lost their viability more than the healthy ones. In conclusion, laser therapy is a viable method to destroy the target cells and has a minimum effect on the healthy tissues and cells and it can improve the other method of cancer treatments limitations.

Keywords: breast cancer, electrospun scaffold, polycaprolacton, laser diode, cancer treatment

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Authors: Milica B. Veljković, Milica B. Simović, Marija M. Ćorović, Ana D. Milivojević, Anja I. Petrov, Katarina M. Banjanac, Dejan I. Bezbradica

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In recent decades, the scientific community has recognized the growing importance of prebiotics, and therefore, numerous studies are focused on their economic production due to their low presence in natural resources. It has been confirmed that prebiotics is a source of energy for probiotics in the gastrointestinal tract (GIT) and enable their proliferation, consequently leading to the normal functioning of the intestinal microbiota. Also, products of their fermentation are short-chain fatty acids (SCFA), which play a key role in maintaining and improving the health not only of the GIT but also of the whole organism. Among several confirmed prebiotics, fructooligosaccharides (FOS) are considered interesting candidates for use in a wide range of products in the food industry. They are characterized as low-calorie and non-cariogenic substances that represent an adequate sugar substitute and can be considered suitable for use in products intended for diabetics. The subject of this research will be the production of FOS by transforming sucrose using a fructosyltransferase (FTase) present in commercial preparation Pectinex® Ultra SP-L, with special emphasis on the development of adequate FTase immobilization method that would enable selective isolation of the enzyme responsible for the synthesis of FOS from the complex enzymatic mixture. This would lead to considerable enzyme purification and allow its direct incorporation into different sucrose-based products without the fear that the action of the other hydrolytic enzymes may adversely affect the products' functional characteristics. Accordingly, the possibility of selective immobilization of the enzyme using support with primary amino groups, Purolite® A109, which was previously activated and modified using glutaraldehyde (GA), was investigated. In the initial phase of the research, the effects of individual immobilization parameters such as pH, enzyme concentration, and immobilization time were investigated to optimize the process using support chemically activated with 15% and 0.5% GA to form dimers and monomers, respectively. It was determined that highly active immobilized preparations (371.8 IU/g of support - dimer and 213.8 IU/g of support – monomer) were achieved under acidic conditions (pH 4) provided that an enzyme concentration was 50 mg/g of support after 7 h and 3 h, respectively. Bearing in mind the obtained results of the expressed activity, it is noticeable that the formation of dimers showed higher reactivity compared to the form of monomers. Also, in the case of support modification using 15% GA, the value of the ratio of FTase and pectinase (as dominant enzyme mixture component) activity immobilization yields was 16.45, indicating the high feasibility of selective immobilization of FTase on modified polystyrene resin. After obtaining immobilized preparations of satisfactory features, they were tested in a reaction of FOS synthesis under determined optimal conditions. The maximum FOS yields of approximately 50% of total carbohydrates in the reaction mixture were recorded after 21 h. Finally, it can be concluded that the examined immobilization method yielded highly active, stable and, more importantly, refined enzyme preparation that can be further utilized on a larger scale for the development of continual processes for FOS synthesis, as well as for modification of different sucrose-based mediums.

Keywords: chemical modification, fructooligosaccharides, glutaraldehyde, immobilization of fructosyltransferase

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Authors: E. S. Johal, M. S. Saini, M. K. Jha

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Currently, 1.1 billion people are at risk due to lack of clean water and about 35 % of people in the developed world die from water related problem. To alleviate these problems water purification technology requires new approaches for effective management and conservation of water resources. Electrospun nanofibres membrane has a potential for water purification due to its high large surface area and good mechanical strength. In the present study PAMAM dendrimers composite nynlon-6 nanofibres membrane was prepared by crosslinking method using Glutaraldehyde. Further, the efficacy of the modified membrane can be renewed by mere exposure of the saturated membrane with the solution having acidic pH. The modified membrane can be used as an effective tool for water purification.

Keywords: dendrimer, nanofibers, nanocomposite membrane, water purification

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Authors: Mahmoud H. Abu Elella, Riham R. Mohamed, Magdy W. Sabaa

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Firstly, synthesis of N-Quaternized Chitosan (NQC), then it was proven by FTIR and 1H-NMR analysis. The degree of quaternization(DQ 35% ) was determined by equation. Secondly, synthesis of cross-linked hydrogels composed of NQC and poly (vinyl alcohol) (PVA) in different weight ratios in presence of glutaraldehyde (GA) as cross-linking agent. Characterization of the prepared hydrogels was done using FTIR, SEM, XRD,and TGA. Swellability in simulated body fluid (SBF) solutions applied on NQC / PVA hydrogels and swelling rate(Wt%) and metal ions uptake was done on it.

Keywords: hydrogel, metal ions uptake, N-quaternized chitosan, poly (vinyl alcohol), swellability

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Authors: Francisco J. Cedano, Laura M. Pinzón, Camila I. Castro, Felipe Salcedo, Juan P. Casas, Juan C. Briceño

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Complex fractures located in articular surfaces are challenging to treat and their reduction with conventional treatments could compromise the functionality of the affected limb. An adhesive material to treat those fractures is desirable for orthopedic surgeons. This adhesive must be biocompatible and have a high adhesion to bone surface in an aqueous environment. The proposed adhesive is based on chitosan, given its adhesive and biocompatibility properties. Chitosan is mixed with calcium carbonate and hydroxyapatite, which contribute to structural support and a gel like behavior, and glutaraldehyde is used as a cross-linking agent to keep the adhesive mechanical performance in aqueous environment. This work aims to evaluate the rheological, adhesion strength and biocompatibility properties of the proposed adhesive using in vitro tests. The gelification process of the adhesive was monitored by oscillatory rheometry in an ARG-2 TA Instruments rheometer, using a parallel plate geometry of 22 mm and a gap of 1 mm. Time sweep experiments were conducted at 1 Hz frequency, 1% strain and 37°C from 0 to 2400 s. Adhesion strength is measured using a butt joint test with bovine cancellous bone fragments as substrates. The test is conducted at 5 min, 20min and 24 hours after curing the adhesive under water at 37°C. Biocompatibility is evaluated by a cytotoxicity test in a fibroblast cell culture using MTT assay and SEM. Rheological results concluded that the average gelification time of the adhesive is 820±107 s, also it reaches storage modulus magnitudes up to 106 Pa; The adhesive show solid-like behavior. Butt joint test showed 28.6 ± 9.2 kPa of tensile bond strength for the adhesive cured for 24 hours. Also there was no significant difference in adhesion strength between 20 minutes and 24 hours. MTT showed 70 ± 23 % of active cells at sixth day of culture, this percentage is estimated respect to a positive control (only cells with culture medium and bovine serum). High vacuum SEM observation permitted to localize and study the morphology of fibroblasts presented in the adhesive. All captured fibroblasts presented in SEM typical flatted structure with filopodia growth attached to adhesive surface. This project reports an adhesive based on chitosan that is biocompatible due to high active cells presented in MTT test and these results were correlated using SEM. Also, it has adhesion properties in conditions that model the clinical application, and the adhesion strength do not decrease between 5 minutes and 24 hours.

Keywords: bioadhesive, bone adhesive, calcium carbonate, chitosan, hydroxyapatite, glutaraldehyde

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Authors: Mirjana Petronijević, Sanja Panić, Aleksandra Cvetanović, Branko Kordić, Nenad Grba

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Enzyme peroxidases are biological catalysts and play a major role in phenolic wastewater treatments and other environmental applications. The most studied species from the peroxidases family is horseradish peroxidase (HRP). In environmental processes, HRP could be used in its free or immobilized form. Enzyme immobilization onto solid support is performed to improve the enzyme properties, prolong its lifespan and operational stability and allow its reuse in industrial applications. One of the enzyme supports of a newer generation is magnetic particles (MPs). Fe₃O₄ MPs are the most widely pursued immobilization of enzymes owing to their remarkable advantages of biocompatibility and non-toxicity. Also, MPs can be easily separated and recovered from the water by applying an external magnetic field. On the other hand, metals and metal oxides are not suitable for the covalent binding of enzymes, so it is necessary to perform their surface modification. Fe₃O₄ MPs functionalization could be performed during the process of their synthesis if it takes place in the presence of plant extracts. Extracts of plant material, such as wild plants, herbs, even waste materials of the food and agricultural industry (bark, shell, leaves, peel), are rich in various bioactive components such as polyphenols, flavonoids, sugars, etc. When the synthesis of magnetite is performed in the presence of plant extracts, bioactive components are incorporated into the surface of the magnetite, thereby affecting its functionalization. In this paper, the suitability of bio-magnetite as solid support for covalent immobilization of HRP across glutaraldehyde was examined. The activity of immobilized HRP at different pH values (4-9) and temperatures (20-80°C) and reusability were examined. Bio-MP was synthesized by co-precipitation method from Fe(II) and Fe(III) sulfate salts in the presence of water extract of the Allium cepa peel. The water extract showed 81% of antiradical potential (according to DPPH assay), which is connected with the high content of polyphenols. According to the FTIR analysis, the bio-magnetite contains oxygen functional groups (-OH, -COOH, C=O) suitable for binding to glutaraldehyde, after which the enzyme is covalently immobilized. The immobilized enzyme showed high activity at ambient temperature and pH 7 (30 U/g) and retained ≥ 80% of its activity at a wide range of pH (5-8) and temperature (20-50°C). The HRP immobilized onto bio-MPs showed remarkable stability towards temperature and pH variations compared to the free enzyme form. On the other hand, immobilized HRP showed low reusability after the first washing cycle enzyme retains 50% of its activity, while after the third washing cycle retains only 22%.

Keywords: bio-magnetite, enzyme immobilization, water extracts, environmental protection

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Authors: Areej K. Al-Jwaid, Dmitiry Berllio, Andrew Cundy, Irina Savina, Jonathan L. Caplin

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Phenol is a toxic compound that is widely distributed in the environment including the atmosphere, water and soil, due to the release of effluents from the petrochemical and pharmaceutical industries, coking plants and oil refineries. Moreover, a range of daily products, using phenol as a raw material, may find their way into the environment without prior treatment. The toxicity of phenol effects both human and environment health, and various physio-chemical methods to remediate phenol contamination have been used. While these techniques are effective, their complexity and high cost had led to search for alternative strategies to reduce and eliminate high concentrations of phenolic compounds in the environment. Biological treatments are preferable because they are environmentally friendly and cheaper than physico-chemical approaches. Some microorganisms such as Pseudomonas sp., Rhodococus sp., Acinetobacter sp. and Bacillus sp. have shown a high ability to degrade phenolic compounds to provide a sole source of energy. Immobilisation process utilising various materials have been used to protect and enhance the viability of cells, and to provide structural support for the bacterial cells. The aim of this study is to develop a new approach to the bioremediation of phenol based on an immobilisation strategy that can be used in wastewater. In this study, two bacterial species known to be phenol degrading bacteria (Pseudomonas mendocina and Rhodococus koreensis) were purchased from National Collection of Industrial, Food and Marine Bacteria (NCIMB). The two species and mixture of them were immobilised to produce macro porous crosslinked cell cryogels samples by using four types of cross-linker polymer solutions in a cryogelation process. The samples were used in a batch culture to degrade phenol at an initial concentration of 50mg/L at pH 7.5±0.3 and a temperature of 30°C. The four types of polymer solution - i. glutaraldehyde (GA), ii. Polyvinyl alcohol with glutaraldehyde (PVA+GA), iii. Polyvinyl alcohol–aldehyde (PVA-al) and iv. Polyetheleneimine–aldehyde (PEI-al), were used at different concentrations, ranging from 0.5 to 1.5% to crosslink the cells. The results of SEM and rheology analysis indicated that cell-cryogel samples crosslinked with the four cross-linker polymers formed monolithic macro porous cryogels. The samples were evaluated for their ability to degrade phenol. Macro porous cell–cryogels crosslinked with GA and PVA+GA showed an ability to degrade phenol for only one week, while the other samples crosslinked with a combination of PVA-al + PEI-al at two different concentrations have shown higher stability and viability to reuse to degrade phenol at concentration (50 mg/L) for five weeks. The initial results of using crosslinked cell cryogel samples to degrade phenol indicate that is a promising tool for bioremediation strategies especially to eliminate and remove the high concentration of phenol in wastewater.

Keywords: bioremediation, crosslinked cells, immobilisation, phenol degradation

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Authors: Rafeqah Raslan, Mastura AbdManaf, Junaidah Jai, Istikamah Subuki, Ana Najwa Mustapa

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The volatile citronella essential oil was encapsulated by simple coacervation and complex coacervation using gum Arabic and gelatin as wall material. Glutaraldehyde was used in the methodology as crosslinking agent. The citronella standard calibration graph was developed with R2 equal to 0.9523 for the accurate determination of encapsulation efficiency and release study. The release kinetic was analyzed based on Fick’s law of diffusion for polymeric system and linear graph of log fraction release over log time was constructed to determine the release rate constant, k and diffusion coefficient, n. Both coacervation methods in the present study produce encapsulation efficiency around 94%. The capsules morphology analysis supported the release kinetic mechanisms of produced capsules for both coacervation process.

Keywords: simple coacervation, complex coacervation, encapsulation efficiency, release kinetic study

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Authors: S. Mohammadzadehmoghadam, Y. Dong

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In this study, Bombyx mori silk fibroin/gelatin (SF/GT) nanocomposite with different GT ratio (SF/GT 100/0, 90/10 and 70/30) were prepared by electrospinning process and crosslinked with glutaraldehyde (GA) vapor. Properties of crosslinked SF/GT nanocomposites were investigated by scanning electron microscopy (SEM), mechanical test, water uptake capacity (WUC) and porosity. From SEM images, it was found that fiber diameter increased as GT content increased. The results of mechanical test indicated that the SF/GT 70/30 nanocomposites had both the highest Young’s modulus of 342 MPa and the highest tensile strength of about 14 MPa. However, porosity and WUC decreased from 62% and 405% for pristine SF to 47% and 232% for SF/GT 70/30, respectively. This behavior can be related to higher degree of crosslinking as GT ratio increased which altered the structure and physical properties of scaffolds. This study showed that incorporation of GT into SF nanofibers can enhance mechanical properties of resultant nanocomposite, but the GA treatment should be optimized to control and fine-tune other properties to warrant their biomedical application.

Keywords: electrospinning, gelatin, silk fibroin, mechanical properties, nanocomposites

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Authors: Chih-Chang Yeh, Min-Fang Yang, Hsin-I Chang

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Chitosan is a cationic polysaccharide derived from the partial deacetylation of chitin. Hyaluronic acid (HA), chondroitin sulfate (CS) and heparin (HP) are anionic glycosaminoglycans (GCGs) which can regulate osteogenic activity. In this study, chitosan membranes were prepared by glutaraldehyde crosslinking reaction and then complexed with three different types of GCGs. 7F2 osteoblasts-like cells and macrophages Raw264.7 were used as models to study the influence of chitosan membranes on osteometabolism. Although chitosan membranes are highly hydrophilic, the membranes associated with GCG-chitosan complexes showed about 60-70% cell attachment. Furthermore, the membranes associated with HP-chitosan complexes could increase ALP activity in comparison with chitosan films only. Three types of the membranes associated with GCG-chitosan complexes could significantly inhibit LPS induced-nitric oxide expression. In addition, chitosan membranes associated with HP and HA can down-regulate tartrate-resistant acid phosphatase (TRAP) activity but not CS-chitosan complexes. Based on these results, we conclude that chitosan membranes associated with HP can increase ALP activity in osteoblasts and chitosan membranes associated with HP and HA reduce TRAP activity in osteoclasts.

Keywords: osteoblast, osteoclast, chitosan, glycosaminoglycan

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Authors: M. Cynthya, V. Prabhawathi, D. Mukesh

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Food contamination occurs during post process handling. This leads to spoilage and growth of pathogenic microorganisms in the food, thereby reducing its shelf life or spreading of food borne diseases. Several methods are tried and one of which is use of antimicrobial packaging. Here, papain, a protease enzyme, is covalently immobilized with the help of glutarldehyde on polyurethane and used as a food wrap to protect food from microbial contamination. Covalent immobilization of papain was achieved at a pH of 7.4; temperature of 4°C; glutaraldehyde concentration of 0.5%; incubation time of 24 h; and 50 mg of papain. The formation of -C=N- observed in the Fourier transform infrared spectrum confirmed the immobilization of the enzyme on the polymer. Immobilized enzyme retained higher activity than the native free enzyme. The efficacy of this was studied by wrapping it over S. aureus contaminated cottage cheese (paneer) and cheese and stored at a temperature of 4°C for 7 days. The modified film reduced the bacterial contamination by eight folds when compared to the bare film. FTIR also indicates reduction in lipids, sugars and proteins in the biofilm.

Keywords: cheese, papain, polyurethane, Staphylococcus aureus

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Authors: Tian-Fang Kang, Chao-Nan Ge, Rui Li

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An electrochemical biosensor for the determination of organophosphorus pesticides was developed based on electrochemical co-deposition of Au and Pd nanoparticles on glassy carbon electrode (GCE). Energy disperse spectroscopy (EDS) analysis was used for characterization of the surface structure. Scanning electron micrograph (SEM) demonstrates that the films are uniform and the nanoclusters are homogeneously distributed on the GCE surface. Acetylcholinesterase (AChE) was immobilized on the Au and Pd nanoparticle modified electrode (Au-Pd/GCE) by cross-linking with glutaraldehyde. The electrochemical behavior of thiocholine at the biosensor (AChE/Au-Pd/GCE) was studied. The biosensors exhibited substantial electrocatalytic effect on the oxidation of thiocholine. The peak current of linear scan voltammetry (LSV) of thiocholine at the biosensor is proportional to the concentration of acetylthiocholine chloride (ATCl) over the range of 2.5 × 10-6 to 2.5 × 10-4 M in 0.1 M phosphate buffer solution (pH 7.0). The percent inhibition of acetylcholinesterase was proportional to the logarithm of parathion concentration in the range of 4.0 × 10-9 to 1.0 × 10-6 M. The detection limit of parathion was 2.6 × 10-9 M. The proposed method exhibited high sensitivity and good reproducibility.

Keywords: acetylcholinesterase, Au-Pd nanoparticles, electrochemical biosensors, parathion

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Authors: J. Desbrieres, M. Popa, C. Peptu, S. Bacaita

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Because of their favorable properties (non-toxicity, biodegradability, mucoadhesivity etc.), polysaccharides were studied as biomaterials and as pharmaceutical excipients in drug formulations. These formulations may be produced in a wide variety of forms including hydrogels, hydrogel based particles (or capsules), films etc. In these formulations, the polysaccharide based materials are able to provide local delivery of loaded therapeutic agents but their delivery can be rapid and not easily time-controllable due to, particularly, the burst effect. This leads to a loss in drug efficiency and lifetime. To overcome the consequences of burst effect, systems involving liposomes incorporated into polysaccharide hydrogels may appear as a promising material in tissue engineering, regenerative medicine and drug loading systems. Liposomes are spherical self-closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent into their structure. The simplicity of production, their biocompatibility, the size and similar composition of cells, the possibility of size adjustment for specific applications, the ability of hydrophilic or/and hydrophobic drug loading make them a revolutionary tool in nanomedicine and biomedical domain. Drug delivery systems were developed as hydrogels containing chitosan or carboxymethylcellulose (CMC) as polysaccharides and gelatin (GEL) as polypeptide, and phosphatidylcholine or phosphatidylcholine/cholesterol liposomes able to accurately control this delivery, without any burst effect. Hydrogels based on CMC were covalently crosslinked using glutaraldehyde, whereas chitosan based hydrogels were double crosslinked (ionically using sodium tripolyphosphate or sodium sulphate and covalently using glutaraldehyde). It has been proven that the liposome integrity is highly protected during the crosslinking procedure for the formation of the film network. Calcein was used as model active matter for delivery experiments. Multi-Lamellar vesicles (MLV) and Small Uni-Lamellar Vesicles (SUV) were prepared and compared. The liposomes are well distributed throughout the whole area of the film, and the vesicle distribution is equivalent (for both types of liposomes evaluated) on the film surface as well as deeper (100 microns) in the film matrix. An obvious decrease of the burst effect was observed in presence of liposomes as well as a uniform increase of calcein release that continues even at large time scales. Liposomes act as an extra barrier for calcein release. Systems containing MLVs release higher amounts of calcein compared to systems containing SUVs, although these liposomes are more stable in the matrix and diffuse with difficulty. This difference comes from the higher quantity of calcein present within the MLV in relation with their size. Modeling of release kinetics curves was performed and the release of hydrophilic drugs may be described by a multi-scale mechanism characterized by four distinct phases, each of them being characterized by a different kinetics model (Higuchi equation, Korsmeyer-Peppas model etc.). Knowledge of such models will be a very interesting tool for designing new formulations for tissue engineering, regenerative medicine and drug delivery systems.

Keywords: controlled and delayed release, hydrogels, liposomes, polysaccharides

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Authors: S. H. Rashmi, G. M. Madhu, A. A. Kittur, R. Suresh

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Polymer nanocomposites represent a new class of materials in which nanomaterials act as the reinforcing material in composites, wherein small additions of nanomaterials lead to large enhancements in thermal, optical, and mechanical properties. A boost in these properties is due to the large interfacial area per unit volume or weight of the nanoparticles and the interactions between the particle and the polymer. Micro-sized particles used as reinforcing agents scatter light, thus, reducing light transmittance and optical clarity. Efficient nanoparticle dispersion combined with good polymer–particle interfacial adhesion eliminates scattering and allows the exciting possibility of developing strong yet transparent films, coatings and membranes. This paper aims at synthesizing zinc oxide nanoparticles which are reinforced in poly vinyl alcohol (PVA) polymer. The mechanical properties showed that the tensile strength of the PVA nanocomposites increases with the increase in the amount of nanoparticles.

Keywords: glutaraldehyde, polymer nanocomposites, poly vinyl alcohol, zinc oxide

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Authors: Lie-Sian Yap, Ming-Chien Yang

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This study demonstrated a novel injectable hydrogel scaffold composing of Pluronic F127, carboxymethyl hexanoyl chitosan (CA) and glutaraldehyde (GA) for encapsulating human fetal osteoblastic cells (hFOB) 1.19. The hydrogel was prepared by mixing F127 and GA in CA solution at 4°C. The mechanical properties and cytotoxicity of this hydrogel were determined through rheological measurements and MTT assay, respectively. After encapsulation process, the hFOB 1.19 cells morphology was examined using fluorescent and confocal imaging. The results indicated that the Tgel of this system was around 30°C, where sol-gel transformation occurred within 90s and F127/CA/GA gel was able to remain intact in the medium for more than 1 month. In vitro cell culture assay revealed that F127/CA/GA hydrogels were non-cytotoxic. Encapsulated hFOB 1.19 cells not only showed the spherical shape and formed colonies, but also reduced their size. Moreover, the hFOB 1.19 cells showed that cells remain alive after the encapsulation process. Based on these results, these F127/CA/GA hydrogels can be used to encapsulate cells for tissue engineering applications.

Keywords: carboxymethyl hexanoyl chitosan, cell encapsulation, hFOB 1.19, Pluronic F127

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

Procedia PDF Downloads 195

Authors: S. Md. Shaarani, J. Md. Jahim, R. A. Rahman, R. Md. Illias

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Modern developments in biotechnology have paved the way for extensive use of biocatalysis in industries. Although it offers immense potential, industrial application is usually hampered by lack of operational stability, difficulty in recovery as well as limited re-use of the enzyme. These drawbacks, however, can be overcome by immobilization. Cross-linked enzyme aggregates (CLEAs), a versatile carrier-free immobilization technique is one that is currently capturing global interest. This approach involves precipitating soluble enzyme with an appropriate precipitant and subsequent crosslinking by a crosslinking reagent. Without ineffective carriers, CLEAs offer high enzymatic activity, stability and reduced production cost. This study demonstrated successful CLEA synthesis of recombinant xylanase from Trichoderma reesei using ethanol as aggregating agent and glutaraldehyde (2% (v/v); 100 mM) as crosslinker. Effects of additives including proteic feeder such as bovine serum albumin (BSA) and poly-L-Lysine were investigated to reveal its significance in enhancing the performance of enzyme. Addition of 0.1 mg BSA/U xylanase showed considerable increment in CLEA development with approximately 50% retained activity.

Keywords: cross-linked, immobilization, recombinant, xylanase

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Authors: Nor Khaizan Anuar, Nur Karimah Aziz, Tin Wui Wong, Ahmad Sazali Hamzah, Wan Rozita Wan Engah

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The physicochemical attributes and wound healing activity of pectin hydrogel in rat models, following partial thickness thermal injury were investigated. The pectin hydrogel was prepared by solvent evaporation method with the aid of glutaraldehyde as crosslinking agent and glycerol as plasticizer. The physicochemical properties were mainly evaluated using differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy, while the wound healing activity was examined by the macroscopic images, wound size reduction and histological evaluation using haematoxylin and eosin (H&E) stain for 14 days. The DSC and FTIR analysis suggested that pectin hydrogel exhibited higher extent of polymer-polymer interaction at O-H functional group in comparison to the unprocessed pectin. This was indicated by the increase of endothermic enthalpy values from 139.35 ± 13.06 J/g of unprocessed pectin to 156.23 ± 2.86 J/g of pectin hydrogel, as well as the decrease of FTIR wavenumber corresponding to O-H at 3432.07 ± 0.49 cm-1 of unprocessed pectin to 3412.62 ± 13.06 cm-1 of pectin hydrogel. Rats treated with pectin hydrogel had significantly smaller wound size (Student’s t-test, p<0.05) when compared to the untreated group starting from day 7 until day 14. H&E staining indicated that wounds received pectin hydrogel had more fibroblasts, blood vessels and collagen bundles on day 14 in comparison to the untreated rats.

Keywords: pectin, physicochemical, rats, wound

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Authors: Muhammad Zubair, Aman Ullah, Jianping Wu

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During the last decades, petroleum derived synthetic polymers like polyethylene terephthalate, polyvinylchloride, polyethylene, polypropylene and polystyrene has extensively been used in the field of food packaging and mostly are non-degradable. Biopolymers are a good fit for single-use or short-lived products such as food packaging. Spent hens, a poultry by-product which is of little economic value and their disposal are environmentally harmful. Through current study, we have explored the possibility to transform proteins from spent fowl into green food packaging material. Proteins from spent fowl were extracted within 1 hour using pH shift method with recovery of about 74%. Different plasticizers were tried like glycerol, sorbitol, glutaraldehyde, 1,2 ethylene glycol and 1,2 butanediol. Glycerol was the best plasticizer among all these plasticizers. A naturally occurring and non-toxic cross-linking agent, chitosan, was used to form the chitosan/glycerol/protein blend by casting and compression molding techniques. The mechanical properties were characterized using tensile strength analyzer. The nano-reinforcements with homogeneous dispersion of nanoparticles lead to improved physical properties suggesting that these materials have great potential for food packaging applications.

Keywords: differential scanning calorimetry, dynamic mechanical analysis, scanning electron microscopy, spent hen

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Authors: Dmitriy Berillo, Areej K. A. Al-Jwaid, Jonathan L. Caplin, Andrew Cundy, Irina Savina

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Chlorophenols (CPs) are used as a precursor in the production of higher CPs and dyestuffs, and as a preservative. Contamination by CPs of the ground water is located in the range from 0.15-100mg/L. The EU has set maximum concentration limits for pesticides and their degradation products of 0.1μg/L and 0.5μg/L, respectively. People working in industries which produce textiles, leather products, domestic preservatives, and petrochemicals are most heavily exposed to CPs. The International Agency for Research on Cancers categorized CPs as potential human carcinogens. Existing multistep water purification processes for CPs such as hydrogenation, ion exchange, liquid-liquid extraction, adsorption by activated carbon, forward and inverse osmosis, electrolysis, sonochemistry, UV irradiation, and chemical oxidation are not always cost effective and can cause the formation of even more toxic or mutagenic derivatives. Bioremediation of CPs derivatives utilizing microorganisms results in 60 to 100% decontamination efficiency and the process is more environmentally-friendly compared with existing physico-chemical methods. Microorganisms immobilized onto a substrate show many advantages over free bacteria systems, such as higher biomass density, higher metabolic activity, and resistance to toxic chemicals. They also enable continuous operation, avoiding the requirement for biomass-liquid separation. The immobilized bacteria can be reused several times, which opens the opportunity for developing cost-effective processes for wastewater treatment. In this study, we develop a bioremediation system for CPs based on macroporous materials, which can be efficiently used for wastewater treatment. Conditions for the preparation of the macroporous material from specific bacterial strains (Pseudomonas mendocina and Rhodococus koreensis) were optimized. The concentration of bacterial cells was kept constant; the difference was only the type of cross-linking agents used e.g. glutaraldehyde, novel polymers, which were utilized at concentrations of 0.5 to 1.5%. SEM images and rheology analysis of the material indicated a monolithic macroporous structure. Phenol was chosen as a model system to optimize the function of the cryogel material and to estimate its enzymatic activity, since it is relatively less toxic and harmful compared to CPs. Several types of macroporous systems comprising live bacteria were prepared. The viability of the cross-linked bacteria was checked using Live/Dead BacLight kit and Laser Scanning Confocal Microscopy, which revealed the presence of viable bacteria with the novel cross-linkers, whereas the control material cross-linked with glutaraldehyde(GA), contained mostly dead cells. The bioreactors based on bacteria were used for phenol degradation in batch mode at an initial concentration of 50mg/L, pH 7.5 and a temperature of 30°C. Bacterial strains cross-linked with GA showed insignificant ability to degrade phenol and for one week only, but a combination of cross-linking agents illustrated higher stability, viability and the possibility to be reused for at least five weeks. Furthermore, conditions for CPs degradation will be optimized, and the chlorophenol degradation rates will be compared to those for phenol. This is a cutting-edge bioremediation approach, which allows the purification of waste water from sustainable compounds without a separation step to remove free planktonic bacteria. Acknowledgments: Dr. Berillo D. A. is very grateful to Individual Fellowship Marie Curie Program for funding of the research.

Keywords: bioremediation, cross-linking agents, cross-linked microbial cell, chlorophenol degradation

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Authors: Refal Hussain, Saifuddin M. Nomanbhay

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Glycerol is a valuable raw material for the production of industrially useful metabolites. Among many promising applications for the use of glycerol is its bioconversion to high value-added compounds, such as bioethanol through microbial fermentation. Bioethanol is an important industrial chemical with emerging potential as a biofuel to replace vanishing fossil fuels. The yield of liquid fuel in this process was greatly influenced by various parameters viz, temperature, pH, glycerol concentration, organic concentration, and agitation speed were considered. The present study was undertaken to investigate optimum parameters for bioethanol production from raw glycerol by immobilized mutant Escherichia coli (E.coli) (ATCC11505) strain on chitosan cross linked glutaraldehyde optimized by Taguchi statistical method in shake flasks. The initial parameters were set each at four levels and the orthogonal array layout of L16 (45) conducted. The important controlling parameters for optimized the operational fermentation was temperature 38 °C, medium pH 6.5, initial glycerol concentration (250 g/l), and organic source concentration (5 g/l). Fermentation with optimized parameters was carried out in a custom fabricated shake flask. The predicted value of bioethanol production under optimized conditions was (118.13 g/l). Immobilized cells are mainly used for economic benefits of continuous production or repeated use in continuous as well as in batch mode.

Keywords: bioethanol, Escherichia coli, immobilization, optimization

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Authors: Chao Ding, Jun Shi, Huiping Deng

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This study reports the preparation of a new type yeast-chitosan bio-microcapsule coating sodium alginate and chitosan, with good biocompatibility and mechanical strength. Focusing on the optimum preparation conditions of bio-microcapsule, a dynamic test of yeast-chitosan bio-microcapsule combined with ultrafiltration membrane was established to evaluate both the removal efficiency of major pollutants from raw water and the applicability of this system. The results of orthogonal experiments showed that the optimum preparation procedure are as follows: mix sodium alginate solution (3%) with bacteria liquid in specific proportion, drop in calcium chloride solution (4%) and solidify for 30 min; put the plastic beads into chitosan liquid (1.8%) to overlay film for 10 min and then into glutaraldehyde solution (1%) to get cross-linked for 5 min. In dynamic test, the microcapsules were effective as soon as were added in the system, without any start-up time. The removal efficiency of turbidity, ammonia nitrogen and organic matter was 60%, 80%, and 40%. Besides, the bio-microcapsules were prospective adsorbent for heavy metal; they adsorb Pb and Cr⁶⁺ in water while maintaining high biological activity to degrade ammonia nitrogen and small molecular organics through assimilation. With the presence of bio-microcapsules, the internal yeast strains’ adaptability on the external environment and resistance ability on toxic pollutants will be increased.

Keywords: ammonia nitrogen, bio-microcapsules, ultrafiltration membrane, yeast-chitosan

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