Extraction of dermatan sulfate using ionic liquid-assisted enzymatic digestion: An efficient approach.

Dermatan sulfate is one of the major glycosaminoglycan (GAG) present in the animal hides, which is a waste/byproduct from meat industry. Efficient utilization of these meat industry wastes is garnering attention because these wastes render a possibility for their conversion into useful products. With the increased concerns over health, various initiatives have been developed to permit more efficient utilization of these by-products and thereby directly impacting environmental sustainability. Herein, we demonstrate for the first time an efficient and environmentally safe ionic liquid-assisted enzymatic process for the extraction of dermatan sulfate from buffalo hides. Dermatan sulfate has been extracted, separated, and purified from the GAG mixture using IL-assisted enzymatic digestions and chromatographic separations. NMR, FT-IR, and ESI-MS measurements showed typical characteristic peaks for dermatan sulfate. The advantages of this eco-friendly process adopted include i) use of fewer chemicals, ii) elimination of harsh chemicals, iii) elimination of various steps and sub-steps, iv) reduction in process time (12 h), and v) increase in extraction yield by 75% when compared to conventional enzymatic process (57%). Thus, the use of ionic liquids alongside enzymes will serve as an efficient methodology for the futuristic development of these derived GAGs for their potential applications.

Melamine-Based Polymeric Crosslinker for Cleaner Leather Production.

To augment sustainable tanning, less chrome input, high functional quality leather processed via no restricted substance in processing, and ease to treat the inevitable protein waste generated are the key challenge, and currently, they have become the active part of leather research. Our work covers the synthesis of a formaldehyde-free chromium-incorporated polymeric tanning agent (FF-CIPTA) and its application in a reformed leather processing route which ensures near zero discharge of chromium containing solid waste. The preliminary characterization of FF-CIPTA reveals that the developed product is stable up to pH 5.2, and the particle size distribution ranges from 955 to 1450 nm with 12% Cr2O3 content. The present work significantly reduces the tanning agent input without compromising the thermal stability (103 °C) of the leather because of its multicrosslinking nature. Since the product exhibits a polymeric character, it provides tanning-cum-filling action which in turn reduces the retanning agent consumption in subsequent processes. Scanning electron microscopic study, porosity analysis, and hand assessment results clearly indicate the significant improvement in organoleptic properties. In addition, the process also enjoys the benefits of zero chromium containing solid waste generation, 71.4% reduction in chromium input, and high chromium transfer efficiency (92%) than the conventional process (36%), and 74.4% reduction in total dissolved solids generation. Furthermore, the water consumption and chemical input are reduced by 51.6 and 17%, respectively. Reduction in wastewater treatment cost and a high economic value of chromium-free leather scraps leads to a cumulative gain of US$ 39.84 per ton of raw material processing. Overall, a potential and practical applicability for cleaner and sustainable tanning is well established.

Chromium containing leather trimmings valorization: Sustainable sound absorber from collagen hydrolysate intercalated electrospun nanofibers.

Developing value-added material from industrial waste is one of the sustainable ways of recycling solid waste produced from the leather industry. Noise which makes a considerable negative impact in the day to day life of people needs immediate attention where the sound absorbers play a vital role. Nanofibers can be used as sound absorbers due to their properties like porosity and high surface area. In this study, collagen hydrolysate extracted from waste leather trimmings was utilized to produce multilayer hybrid sound-absorbing material. Collagen hydrolysate was electrospun along with polyvinyl alcohol (PVA) and the layer was sandwiched between polyacrylonitrile (PAN) nanofibrous layers. The hierarchical structure of the composite is more porous on outer layers than medium porous inner collagen hydrolysate- PVA layer. The hybrid material was characterized using various experimental techniques and the sound absorption was measured using two-microphone impedance tube method. From acoustic measurements, it was revealed that the composite showed improved sound absorption in the frequency range of 800-2500 Hz due to its varying pore size. Hence, the leather trimmings as a component of sound-absorbing material creates an innovative solution for discarded leather waste and they can be used in practical applications like room acoustics.

Adsorption of Proteins on Dual Loaded Silica Nanocapsules.

The design of nanocarriers containing hydrophobic and hydrophilic compounds represents a powerful tool for cocktail delivery. Water-in-oil-in-water emulsions constitute an attractive approach, as they offer dual encapsulation and provide a template for the constitution of a capsule. A limitation in the preparation of nano double emulsions is their instability resulting from high curvature radii. In this work, silica nanocapsules (NCs) stable over several months were synthesized. This was achieved by exploiting a double emulsion in which the oil phase is constituted by a combination of oils presenting several volatilities. The decrease of oil droplet size by evaporation favored the deposition of a silica layer at the nanoscale interface. The release of the payload obtained by drying the capsules was investigated by fluorescence spectroscopy. Understanding the interactions between proteins and nanocapsules is a fundamental point for many biological applications. Nanocapsules were exposed to two model proteins, which were bovine serum albumin (BSA) and lysozyme (Ly). These proteins, presenting differences in charges and size, showed distinctive arrangements onto the nanocapsules. Moreover, we have studied changes in α-helix and ß-sheet content, which divulged the interactions between the proteins and the nanocapsules.

Inorganic apatite nanomaterial: Modified surface phenomena and its role in developing collagen based polymeric bio-composite (Coll-PLGA/HAp) for biological applications.

Nano sized bio-composites containing inorganic particles conjugated with polymer and protein are considered as potential material for tissue engineering systems like bone repair and advanced drug delivery. More specifically, hydroxyapatite (HAp), a well known as the strong bioactive material has limitations on reactivity towards biological systems. Thus, this work explains the interaction betweena natural biomaterial Collagen and poly (lactide co-glycolide)-Hydroxyapatite (HAp) composite. PLGA/HAp composite was fabricated by in-situ polymerization of DL-lactide, glycolide and HAp nanoparticles. The prepared PLGA/HAp composite was examined for physico-chemical properties by FTIR, DSC, SEM, and DLS. The microscopic image confirms the positioning of a highly ordered structure containing Coll-PLGA/HAp that leads to enhancement in thermal stability of collagen. The nature of bonding and structural orientation of bio-composite was thoroughly investigated by FTIR and SEM. Toxicity of bio-composites on A549 human lung cancer cell line and L929 mouse normal cell line were analysed, and results showed a decreasing trend in the cell viability, on increasing the concentration of bio-composite. As an effective option for tissue engineering, the scaffold was prepared by vacuum drying method. Porosity and tensile strength measurements of scaffold reveal that non-toxic characteristics of bio-composite, excellent pore distribution of scaffold and thermal resistivity make it a versatile material for tissue engineering.

Is Cr(III) toxic to bacteria: toxicity studies using Bacillus subtilis and Escherichia coli as model organism.

The objective of this work is to detect the mode of damage caused by Cr(III), one of the widely used industrial pollutant on Bacillus subtilis-industrial strain 168 and Escherichia coli MTCC 40. Bioassays are very sensitive, precise, economical and rapid for detecting early stages of pollution. The detrimental effect of trivalent chromium becomes clear from the growth profile and growth inhibition studies. Mode of action of damage by trivalent chromium in bacterial model was found to be oxidative, as chromium is one of the redox active metals. The generation of reactive oxygen species (ROS) resulted in membrane damage which in turn had a detrimental effect on the membrane proteins as well as the DNA. The structural changes in the SEM and AFM images clearly reveals the damage caused by Cr(III) to the test bacterial models. Trivalent chromium causes greater DNA, protein and membrane damage in case of E. coli than B. subtilis. Membrane damage caused by ROS becomes evident from the production of Thiobarbituric acid reactive substances (TBARs) as the mechanism of killing followed by DNA damage and the production of elevated levels of stress proteins known as extracellular cellular proteins.

Differential behavior of native and denatured collagen in the presence of alcoholic solvents: A gateway to instant structural analysis.

We report a novel method that exploits the differential solubility properties of native and denatured proteins to distinguish between them. We chose to study this using collagen because its unique native triple helical structure is critical for the desired properties of collagen-based biomaterials. We found that native and denatured collagen separate out from solution in alcohols(methanol, ethanol, n-propanol and isopropanol), but as different phases. Native collagen undergoes tight gel-like separation(macrophase) from the solution in alcohols(90%,v/v), whereas denatured collagen separates as particles(microphase), which turn milky-like turbid. It was found that the pH of the medium had to be alkaline for turbidity formation, which necessitated the use of buffered alcohols. In solutions having mixtures of native and denatured collagen, the method has sensitivity to visually confirm the presence of native collagen even as low as 10%. It was also confirmed that the formation of turbidity was a direct function of the concentration of denatured collagen. A thermal denaturation experiment, wherein stages of denaturation were studied both by the proposed method and circular dichroism, showed that information obtained from both methods of analysis was comparable. This highlights the potential of the proposed method to become an instant fool proof test for collagen.

Selective toxicity of Catechin-a natural flavonoid towards bacteria.

Catechin is a plant polyphenol composed of epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) as diastereoisomers. Among the various classes of flavonoids, catechin was found to be the most powerful free radical scavenger, scavenging the reactive oxygen species (ROS) generated due to oxidative damage with antibacterial and anti-inflammatory activity. The toxicity of catechin towards bacteria was studied using gram-positive bacteria (B. subtilis) and gram-negative bacteria (E. coli) as model organisms and was found to be more toxic towards gram-positive bacteria. From the results, catechin was found to be beneficial as well as toxic (inhibitory) to the bacteria at a selective concentration behaving as double-edged swords with an IC50 value of 9 ppm for both the bacteria. The inhibitory mechanism of catechin was by oxidative damage through membrane permeabilization which was confirmed by the formation and treatment of bacterial liposomes. SEM images of the control and treated bacteria reveals membrane damage with morphological changes.

Dual utility of a novel, copper enhanced laccase from Trichoderma aureoviridae.

Ever since the ability of laccase to oxidize non-phenolic lignin models was described, the oxidative degradation reactions catalyzed by laccase have been widely studied for paper pulp production or detoxification of aromatic pollutants. The viability of developing eco-friendly, laccase aided industrial processes has been explored. Here, we report the isolation and screening of fungi to explore their lignolytic ability on solid media using various substrates as indicators. The promising fungus was cultivated in submerged and solid state conditions. The crude enzyme obtained yielded elevated activity at 75°C and pH 9.0. Addition of CuSO4 increased the activity by almost 25% proving that Cu(2+) catalytically enhances the action of laccases. Decolorization studies were carried out using industrial dye, Remazol Brilliant Blue R (CI 61200) on solid and liquid medium. Visual decolorization was observed within 2 days of inoculation on solid media whereas, liquid medium incorporated with varying concentrations of dye solution showed a final level of decolorization of up to 76%. Bamboo degradation studies revealed a decrease in lignin content by 51 and 43% within a month. To the best of our knowledge, this study for the first time reports that Trichoderma aureoviridae can produce lignolytic enzyme and degrade lignin.

Effect of aqueous ethanol on the triple helical structure of collagen.

Collagen, the most abundant protein in mammals, is widely used for making biomaterials. Recently, organic solvents have been used to fabricate collagen-based biomaterials for biological applications. It is therefore necessary to understand the behavior of collagen in the presence of organic solvents at low (≤50%, v/v) and high (≥90%, v/v) concentrations. This study was conducted to examine how collagen reacts when exposed to low and high concentrations of ethanol, one of the solvents used to make collagen-based biomaterials. Solubility testing indicated that collagen remains in solution at low concentrations (≤50%, v/v) of ethanol but precipitates (gel-like) thereafter, irrespective of the method of addition of ethanol (single shot or gradual addition); this behavior is different from that observed recently with acetonitrile. Collagen retains its triple helix in the presence of ethanol but becomes thermodynamically unstable, with substantially reduced melting temperature, with increasing concentration of ethanol. It was also found that the CD ellipticity at 222 nm, characteristic of the triple-helical structure, does not correlate with the thermal stability of collagen. Time-dependent experiments reveal that the collagen triple helix is kinetically stable in the presence of 0-40% (v/v) ethanol at low temperature (5 °C) but highly unstable in the presence of ethanol at elevated temperature (~34 °C). These results indicate that when ethanol is used to process collagen-based biomaterials, such factors as temperature and duration should be done taking into account, to prevent extensive damage to the triple-helical structure of collagen.

Stabilization of collagen through bioconversion: An insight in protein-protein interaction.

Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein-protein stabilization and discovers paramount application in tissue engineering.

Influence of PCL on the material properties of collagen based biocomposites and in vitro evaluation of drug release.

Formulation of biodegradable collagen-poly-ε-caprolactone (PCL) based biomaterials for the sustained release of insulin is the main objective of the present work. PCL has been employed to modulate the physico-chemical behavior of collagen to control the drug release. Designed formulations were employed to statistically optimize insulin release parameter profile at different collagen to PCL molar ratios. Circular dichroism, thermoporometry, FTIR, impedance and scanning electron microscopy techniques have been employed to investigate the effect of PCL on hydration dynamics of the collagen molecule, which in turn changes the dissolution parameters of the drug from the systems. Drug entrapment efficiency has been found to be maximum for collagen to PCL molar ratio of 1:2 (>90%). In vitro dissolution test reveals that 99% of the drug was released from composite at collagen to PCL molar ratio of 1:3 and 1:4 within 2h, which indicates that hydrophobicity of the matrix results in weak interaction between lipophilic drug and carrier materials. The least burst release was observed for collagen to PCL molar ratio at 1:2 as synergistic interactions between collagen and PCL was maximum at that particular polymer-polymer ratios. The drug release data indicates super case-II transport of drug (n>1.0).

Dielectric behavior of gelatine-glycosaminoglycans blends: an impedance analysis.

The dielectric behavior of the gelatine-GAGs based blend systems has been studied to understand the dynamic behavior of the water at the protein-GAGs interfaces which are relevant for tissue engineering application. Impedance (Z) and phase have been measured as a function of frequencies from 0.01 Hz to 100 kHz. GAGs tunes the ionic charge drift which initiates polarization mechanisms through charge accumulation at structural interfaces and creates conduction currents. The admittance results showed that at high frequency, the conductivity increases with increasing GAGs concentration indicating changes in hydration shell of the gelatine by the GAGs.

Effect of UV irradiation on the physico-chemical properties of iron crosslinked collagen.

Collagen is the main component of connective tissue and finds immense applications as a biomaterial. In this study, effect of UV irradiation on collagen crosslinked with iron has been carried out. The physical and optical properties of crosslinked collagen affected by UV irradiation were analyzed using electrospectral and fluorescence studies. The electronic spectral studies showed that the photoproducts formed on UV radiation decrease in the presence of iron. Circular dichroic studies revealed that the conformational changes brought about in the protein due to UV irradiation have been reduced owing to the crosslinking with iron. However, prolonged irradiation does bring about conformational changes to the protein.

Novel approach towards recovery of glycosaminoglycans from tannery wastewater.

Poly ethylene glycol (PEG)-poly acrylic acid (PAA) based aqueous two-phase system (ATPS) was selected as a practical model to recover glycosaminoglycans (GAGs) from tannery wastewater. The influence of PEG molecular weight, tie line length (TLL), pH, temperature and NaCl concentration on the partition coefficient of glycosaminoglycans from tannery wastewater was studied. Partition coefficient of glycosaminoglycan decreases on increase of PEG molecular weight, NaCl concentration and temperature, whereas it increases with increase of pH. In the PEG-rich phase, increased partitioning of GAGs was observed with increase in TLL. The partitioning of GAGs was better in PEG 4000 at pH 8.0, 20 °C with a yield of 91.50%. This study demonstrates the potential application of ATPS processes for the recovery of GAGs from complex biological suspensions.

Application of a chemically modified green macro alga as a biosorbent for phenol removal.

Phenol and substituted phenols are toxic organic pollutants present in tannery waste streams. Environmental legislation defines the maximum discharge limit to be 5-50 ppm of total phenols in sewers. Thus the efforts to develop new efficient methods to remove phenolic compounds from wastewater are of primary concern. The present work aims at the use of a modified green macro alga (Caulerpa scalpelliformis) as a biosorbent for the removal of phenolic compounds from the post-tanning sectional stream. The effects of initial phenol concentration, contact time, temperature and initial pH of the solution on the biosorption potential of macro algal biomass have been investigated. Biosorption of phenol by modified green macro algae is best described by the Langmuir adsorption isotherm model. Biosorption kinetics of phenol onto modified green macro algal biomass were best described by a pseudo second order model. The maximum uptake capacity was found to be 20 mg of phenol per gram of green macro algae. A Boyd plot confirmed the external mass transfer as the slowest step involved in the biosorption process. The average effective diffusion coefficient was found to be 1.44 x 10(-9) cm(2)/s. Thermodynamic studies confirmed the biosorption process to be exothermic.

Sodium metasilicate based fiber opening for greener leather processing.

Growing environmental regulations propound the need for a transformation in the current practice of leather making. The conventional dehairing and fiber opening process results in high negative impact on the environment because of its uncleanliness. This process accounts for most of the biochemical oxygen demand and chemical oxygen demand in tannery wastewater and generation of H2S gas. Hence, this study explores the use of a biological material and a nontoxic chemical for performing the above process more cleanly. In this study, the dehairing and fiber opening processes has been designed using enzyme and sodium metasilicate. The amount of sodium metasilicate required for fiber opening is standardized through the removal of proteoglycan, increase in weight, and bulk properties of leathers. It has been found that the extent of opening up of fiber bundles is comparable to that of conventionally processed leathers using a 2% sodium metasilicate solution. This has been substantiated through scanning electron microscopic analysis and softness measurements. The presence of silica in the crust leather enhances the bulk properties of the leather. This has been confirmed from the energy dispersive X-ray analysis. Performance of the leathers is shown to be on par with conventionally processed leathers through physical and hand evaluation. The process also exhibits significant reduction in chemical oxygen demand and total solid loads by 55 and 24%, respectively. Further, this newly developed process seems to be economically beneficial.

Dye house wastewater treatment through advanced oxidation process using Cu-exchanged Y zeolite: a heterogeneous catalytic approach.

Catalytic wet hydrogen peroxide oxidation of an anionic dye has been explored in this study. Copper(II) complex of NN'-ethylene bis(salicylidene-aminato) (salenH2) has been encapsulated in super cages of zeolite-Y by flexible ligand method. The catalyst has been characterized by Fourier transforms infra red spectroscopy, X-ray powder diffractograms, Thermo-gravimetric and differential thermal analysis and nitrogen adsorption studies. The effects of various parameters such as pH, catalyst and hydrogen peroxide concentration on the oxidation of dye were studied. The results indicate that complete removal of color has been obtained after a period of less than 1h at 60 degrees C, 0.175M H2O2 and 0.3g l(-1) catalyst. More than 95% dye removal has been achieved using this catalyst for commercial effluent. These studies indicate that copper salen complex encapsulated in zeolite framework is a potential heterogeneous catalyst for removal of color from wastewaters.

Effect of UV irradiation on stabilized collagen: role of chromium(III).

Research on the effect of UV radiation on stabilized collagen is an area of potential interest owing to the fact that collagen is an important biomaterial finding immense use in various fields. In this present study, effect of UV irradiation on collagen stabilized using chromium(III) has been studied. The physical and optical properties affected by UV irradiation have been detailed. Viscosity measurements have shown that chromium(III) treated collagen has better stability against UV radiation than native collagen. Circular dichroic studies indicate that increase in concentration of chromium(III) does not affect the conformation of collagen however, the duration of irradiation has profound impact on the conformation of collagen. The fluorescence intensity of native collagen has been found to decrease more than that of chromium(III) treated collagen. The difference absorption spectra also shows that chromium(III) treatment brings about more stability to collagen against UV irradiation.

Biological removal of carcinogenic chromium(VI) using mixed Pseudomonas strains.

The contamination of soil and wastewaters with Cr(VI) is a major problem. It has been suggested that microbial methods for Cr(VI) reduction are better than chemical methods, as they do not add other ions or toxic chemicals to the environment. In this study an aerobic reduction of Cr(VI) to Cr(III) by employing mixed Pseudomonas cultures isolated from a marshy land has been reported. The role of chromium concentration, temperature, pH and additives on the microbial reduction of Cr(VI) has been investigated. NADH was found to enhance the rate of reduction of Cr(VI). Complete reduction of chromium(VI) has been possible even at chromium(VI) concentrations of 300 ppm. Ions like SO(4)(2-) and poly-phenols inhibited the metabolic activity relating to Cr(VI) reduction. Under optimal conditions 100 mg/L of Cr(VI) was completely reduced within 180 min.