Sandeep Kale

Study of Glycerol and Sweet Water as a Carbon Source for Production of Rhamnolipids by Naturally Occurring Strains of Pseudomonas aeruginosa (ATCC 10145 and ATCC 9027)

Abstract The interest in biosurfactant has increased considerably due to their large potential for industrial applications. Rhamnolipid is a simplest class of biosurfactants with well defined structure. The main obstacle in commercialization of biosurfactant especially rhamnolipids is the higher cost of the production. Glycerol is readily available from commercial fat-splitting process in the form of sweet water (up to 20% w/v) at very low cost. Two natural strains of Pseudomonas aeruginosa (ATCC 10145 and ATCC 9027) were compared for rhamnolipid production. The neotype strain of Pseudomonas aeruginosa (ATCC 10145) was found to produce more amounts of rhamnolipids than Pseudomonas aeruginosa (ATCC 9027) on glucose and glycerol as carbon source. The microbial growth (0.94 g/L) and formation of rhamnolipids (2.75 g/L) was maximum at 5% (w/v) glycerol concentration by Pseudomonas aeruginosa (ATCC 10145). The fermentation was monitored for 8 days on shake flask and magnetically stirred batch culture. In magnetically stirred batch culture, microbial growth was 0.69 g/L and 2.73 g/L of rhamnolipid was formed. Sweet water was also used as a source of glycerol.

Study of Glycerol Residue as a Carbon Source for Production of Rhamnolipids by Pseudomonas aeruginosa (ATCC 10145)

Abstract Rhamnolipid is the simplest class of biosurfactants with a well defined structure. The main obstacle in commercialization of biosurfactant especially rhamnolipids is the higher cost of the production. The production cost can be reduced by using economical carbon sources. Glycerol residue is available from glycerin distillation plant at low cost. It can be used as carbon source for rhamnolipid production using Pseudomonas aeruginosa (ATCC 10145). The optimum concentration of glycerol residue was 5% weight by volume (w/v) yielding maximum rhamnolipids at 2.50 g/L in the broth. From batch monitoring of polyglycerol and monoglycerol consumption, it appeared that polyglycerols were first hydrolyzed to monoglycerol in the broth itself and consumed as carbon source. The product had components similar to rhamnolipids synthesized on pure glycerol under the same conditions. This indicated the potential of glycerol residue as economic substrate for production of rhamnolipids.

Kafirin adsorption on ion-exchange resins: Isotherm and kinetic studies

Kafirin is a natural, hydrophobic and celiac safe prolamin protein obtained from sorghum seeds. Today kafirin is found to be useful in designing delayed delivery systems and coatings of pharmaceuticals and nutraceuticals where its purity is important and this can be obtained by adsorptive chromatography. This study is the first scientific insight into the isotherm and kinetic studies of kafirin adsorption on anion- and cation-exchange resins for practical applications in preparative scale chromatography. Adsorption isotherms of kafirin were determined for five anion- and two cation-exchange resins in batch systems. Isotherm parameters such as maximum binding capacity and dissociation constant were determined from Langmuir isotherm, and adsorptive capacity and affinity constant from Freundlich isotherm. Langmuir isotherm was found to fit the adsorption equilibrium data well. Batch uptake kinetics for kafirin adsorption on these resins was also carried out and critical parameters including the diffusion coefficient, film mass transfer coefficient, and Biot number for film-pore diffusion model were calculated. Both the isotherm and the kinetic parameters were considered for selection of appropriate resin for kafirin purification. UNOsphere Q (78.26 mg/ml) and Toyopearl SP-650M (57.4 mg/ml) were found to offer better kafirin binding capacities and interaction strength with excellent uptake kinetics under moderate operating conditions. With these adsorbents, film diffusion resistance was found to be major governing factor for adsorption (Bi<10 and δ<1). Based on designer objective function, UNOsphere Q was found be best adsorbent for binding of kafirin. The data presented is valuable for designing large scale preparative adsorptive chromatographic kafirin purification systems.

Characterization of superporous cellulose matrix for high-throughput adsorptive purification of lysozyme.

Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100–300 μm, mean pore size: 1–3 μm, pore volume: 57–59%, and bulk density: ∼1,438 kg/m3) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for high‐throughput purification of lysozyme from chicken egg whites extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads‐CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high‐throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m3 h) and specific activity (2.2 × 105 U/mg) were higher than that obtained with traditional particulate resins.

Catalytic metallodendrimers grafted on mesoporous polymethacrylate beads for the regioselective synthesis of β-amino alcohols under solvent-free conditions

A new series of dendritic structures was grafted on epoxy activated mesoporous polymethacrylate beads (Sepabeads EB-EP-400). Grafting with the dendritic structures was followed by loading of metal termini to the chelating groups to form metallodendrimer-grafted catalysts. The catalysts were characterized by UV, FTIR, SEM and elemental analysis. Further, a selected catalyst was used to afford β-amino alcohols in a regioselective manner through nucleophilic opening of an oxirane ring with various aromatic amines under solvent-free, mild reaction conditions. The reactions with the developed catalyst at ambient temperature afforded the corresponding β-amino alcohols in high yields (85–99.5%) with excellent regioselectivity. It was found that the developed metallodendrimer-based catalyst is efficient and recyclable without compromising on activity, selectivity and productivity (4 kg L−1 h−1).

Optimization and monitoring of water soluble substrate for synthesis of mannosylerythritol lipids by Pseudozyma antarctica (ATCC 32657)

Biosurfactants are amphipathic molecules, consisting of hydrophobic and hydrophilic domains, which can partition at the interfaces between different fluid phases, such as oil/water or water/air interfaces. Mannosylerithritol lipids (MELs) are a type of glycolipid. The yeast Candida (Pseudozyma) antarctica (ATCC 32657) secretes an extracellular MEL, with biosurfactant properties, when grown on a water soluble and insoluble substrate, such as glycerine or oils. Value added MEL was biologically synthesized, by using two different types of honey as natural and newer water soluble carbon source. Those types of honey as a carbon source were optimized at different concentration of total carbohydrate present in the honey. Further optimized substrate concentration of honey was monitored for 15 days, for studies of MEL yield and cell growth concentration per day on shake flask batch culture. Surfactant properties, such as surface tension, interfacial tension, foaming, emulsification and wetting of MEL, were observed. Produced MEL was confirmed by thin layer chromatography (TLC), Fourier Transform Infrared (FTIR) spectra, and 1H Nuclear Magnetic Resonance (NMR) techniques.

STUDY OF OLOPATADINE HYDROCHLORIDE UNDER ICH RECOMMENDED STRESS CONDITIONS BY LC, LC-MS/TOF FOR IDENTIFICATION, AND CHARACTERIZATION OF DEGRADATION PRODUCTS

The objective of the present investigation was to separate, identify and characterize the major degradation products of olopatadine hydrochloride, an anti-allergic drug, generated under different stress conditions as per International Conference on Harmonization (ICH) guidelines Q1A (R2). The drug underwent transformation under acidic, basic, and photolytic stress, whereas it was stable in oxidative and thermal stress conditions. Total five degradation products were formed, which were separated by using HPLC on Inertsil ODS 3 (250 mm × 4.6 mm, 5 µm) column using gradient elution program. A complete mass fragmentation pathway of the drug was first established with the help of LC-MS/TOF accurate mass studies. The stress samples were subjected to LC-MS/TOF studies. The obtained mass spectral data were employed to characterize four degradation products. The products formed under degradation study were identified as 11-[(E)-3-(Dimethylamino) propylidene]-6, 11-dihydrodibenz [b, e] oxepin-2-acetic acid, 2-((Z)-9-(3-(dimethylamino) propylidene)-9H-fluoren-7-yl) acetic acid, 11-[(E)-3-(Dimethylamino) propylidene]-6, 11-dihydrodibenz [b, e] oxepin-2-acetic acid methyl ester, 11-[(Z)-3-(Dimethylamino) propylidene]-6, and 11-dihydrodibenz [b, e] oxepin-2-acetic acid methyl ester. The developed method was successfully used for analysis of formulation.

Sophorolipids Synthesized Using Non-Traditional Oils with Glycerol and Studies on Their Surfactant Properties with Synthetic Surfactant

Abstract The importances of bio-surfactants in industrial applications are huge due to their biodegradable and eco-friendly nature. Bio-surfactants mostly find application in cosmetics and health care products. Moreover, bio-surfactants like sophorolipids (SL) also exhibit antimicrobial and skin healing properties. The current studies involve production of SL using low cost substrates like glycerol (15%) instead of glucose (10%) with non-traditional oils (10%) such as jatropha oil, karanja oil and neem oil by using Starmerella bombicola (ATCC 22214). Neem oil gave lower yield i.e. (1.42 g/L) of SL as compared to jatropha oil (4.74 g/L) and karanja oil (5.91 g/L) with glycerol as substrate. Some pretreatment given to crude neem oil like oil refining and ethanol washing with glycerol helps in improved cell growth and SL yield i.e. 2.73 g/L and 3.82 g/L respectively. The comparison thin layer chromatography (TLC), Fourier Transform infrared spectra (FTIR), high performance liquid chromatography (HPLC), liquid chromatography mass spectra (LC-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR) of SL produced on non-traditional oils with glycerol were carried out with that of SL produced on non-traditional oils with glucose. The surfactant properties of sodium lauryl sulphate (SLS) such as surface tension, interfacial tension, stabilization of foam, emulsification, and wetting were improved when SLS was replaced at different concentration of SL.

Co-treatment with sulforaphane–zein microparticles enhances the chemopreventive potential of zinc in a 1,2-dimethylhydrazine induced colon carcinogenesis rat model

Decrease in plasma and tissue zinc is associated with the development of pre-neoplastic lesions, correlating well with the progression to carcinoma in the colon. There is also a decrease in antioxidant proteins like the zinc-dependent metallothionein (MT) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), because of the overwhelming oxidative stress. Sulforaphane (SFN) increases cellular antioxidant capacity by releasing Nrf2 from its inhibitory complex and prevents oxidative damage induced colon carcinogenesis. Administration of zinc also induces synthesis of MT directly and indirectly via its action on Nrf2. Because zinc and SFN both act on MT and Nrf2, we evaluated the effects of co-administration of zinc and SFN formulated as microparticles with zein in a 1,2-dimethylhydrazine (DMH) induced colon carcinogenesis rat model. Groups of rats were sacrificed after the end of four and six weeks and the appearance of non-dysplastic, hyperplastic and dysplastic types of aberrant crypts were considered as biomarkers for evaluating the process of carcinogenesis. Colonic tissue levels of MT and Nrf2 were measured along with other proteins and enzymes to evaluate oxidative stress load in the colonic tissue. The co-treatment showed significantly greater reduction in the formation of aberrant crypts along with increased induction of cellular antioxidant components compared to single treatments. This investigation supports the hypothesis that co-administration of zinc and SFN leads to an enhanced chemopreventive outcomes mainly facilitated by the induction of MT and Nrf2.

Stabilization of Foam Produced by Sodium Lauryl Sulphate with Mannosylerythritol Lipids Synthesized on Soybean Oil and Sucrose by Pseudozyma antarctica (ATCC 32657)

Abstract Biosurfactants are gaining importance due their large potential for industrial applications and their eco-friendly nature. The properties of biosurfactants can vary with their structure which is dependent upon strain, cultivation conditions and carbon source. A mannosylerythritol lipid (MEL) is one of the simplest biosurfactants with a well-defined structure produced by Pseudozyma antarctica (ATCC 32657). The study includes the surfactant properties of MEL produced on soybean oil with sucrose as substrate. MEL showed surface activity at very low concentrations. The various properties were explained on the basis of the structural similarity between MEL and Gemini surfactant. The performance properties of SLS like foaming, emulsification and wetting were improved when SLS was partially substituted (5–20 %) by MEL. Based on the study, MEL can be used in various cosmetic products like shaving creams, skin creams and lotions, where MEL will not only enhance foaming property but also provide moisturizing and healing properties on skin.