Shailendra Raghuwanshi

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde.

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.

Bacillus sphaericus: the highest bacterial tannase producer with potential for gallic acid synthesis.

An indigenously isolated strain of Bacillus sphaericus was found to produce 1.21 IU/ml of tannase under unoptimized conditions. Optimizing the process one variable at a time resulted in the production of 7.6 IU/ml of tannase in 48 h in the presence of 1.5% tannic acid. A 9.26-fold increase in tannase production was achieved upon further optimization using response surface methodology (RSM), a statistical approach. This increase led to a production level of 11.2I U/ml in medium containing 2.0% tannic acid, 2.5% galactose, 0.25% ammonium chloride, and 0.1% MgSO(4) pH 6.0 incubated at 37°C and 100 rpm for 48 h with a 2.0% inoculum level. Scaling up tannase production in a 30-l bioreactor resulted in the production of 16.54 IU/ml after 36 h. Thus far, this tannase production is the highest reported in this bacterial strain. Partially purified tannase exhibited an optimum pH of 5.0 with activity in the pH range of 3 to 8; 50°C was the optimal temperature for activity. Efficient conversion of tannic acid to purified gallic acid (90.80%) was achieved through crystallization.

Evaluation of corncob hemicellulosic hydrolysate for xylitol production by adapted strain of Candida tropicalis.

A maximum xylose extraction of 21.98 g/L was obtained in hydrolysate with a solid to liquid ratio of 1:8 (w/v) at 1% H(2)SO(4) and treated for 30 min. The optimized and treated corncob hemicellulosic hydrolysate medium supplemented with (g/L) yeast extract 5.0, KH(2)PO(4) 2.0, MgSO(4)·7H(2)O 0.3 and methanol 10 mL whose pH was adjusted to 4.5 acts as production medium. Under this condition; the adapted strain of C. tropicalis resulted in 1.22-fold increase in xylitol yield and 1.70-fold enhancement in volumetric productivity was obtained as compared to parent strain of C. tropicalis. On concentrating the hydrolysate under vacuum using rotavapor proves to be efficient in terms of improved xylitol yield and productivity over microwave assisted concentration using adapted strain of C. tropicalis. The immobilized cells of C. tropicalis resulted in more than 70% efficiency up to third cycle. The xylitol production could be scaled up to 10 L fermentor.

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe3O4 nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe3O4-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe3O4-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.

Fermentation behavior of an osmotolerant yeast D. hansenii for Xylitol production

Realizing the importance of xylitol as a high‐valued compound that serves as a sugar substitute, a new, one step thin layer chromatographic procedure for quick, reliable, and efficient determination of xylose and xylitol from their mixture was developed. Two hundred and twenty microorganisms from the laboratory stock cultures were screened for their ability to produce xylitol from D‐xylose. Amongst these, an indigenous yeast isolate no.139 (SM‐139) was selected and identified as Debaryomyces hansenii on the basis of morphological and biochemical characteristics and (26S) D1/D2 r DNA region sequencing. Debaryomyces hansenii produced 9.33 gL−1 of xylitol in presence of 50.0 gL−1 of xylose in 84 h at pH 5.5, 30°C, 200 rpm. In order to utilize even higher concentrations of xylose for maximum xylitol production, a xylose enrichment technique was developed. The strain of Debaryomyces hansenii was obtained through xylose enrichment technique in a statistically optimized medium containing 0.3% yeast extract, 0.2% peptone, 0.03% MgSO4.7H2O along with 1% methanol. The culture was inoculated with 6% inoculum and incubated at 30°C and 250 rpm. A yield of 0.6 gg−1 was obtained with a xylitol volumetric productivity of 0.65 g/L h−1 in the presence of 200 gL−1 of xylose although up to 300 gL−1 of xylose could be tolerated through batch fermentation. Through this technique, even higher concentrations of xylose as substrate could be potentially utilized for maximum xylitol production.

Examine growth inhibition pattern and lactic acid production in Streptococcus mutans using different concentrations of xylitol produced from Candida tropicalis by fermentation.

Twenty clinical isolates of Streptococcus sp. were isolated from six clinical samples of dental caries on MSFA. Amongst these isolates, five clinical isolates were identified as S treptococcus mutans on the basis of morphological, biochemical and 16S rDNA sequencing. The isolated strains of S. mutans were exposed to fermented and purified xylitol (0.25-15.0%) and tested for its anti-microbial effects against control medium (Brain Heart Infusion without xylitol) after 12 h. The plate assay was developed using bromocresol green as an indicator dye in order to study the relative growth inhibition pattern of clinical sample at different concentrations of an anti-microbial compound in a single petriplate. The morphology of S. mutans cells in brain heart infusion (BHI) medium containing xylitol resulted in a diffused cell wall as observed using gram staining technique. The minimum inhibitory concentration (MIC) is 0.25% for S. mutans obtained from different clinical samples. The MIC(50) and MIC(90) is 5.0% and 10.0% xylitol respectively of the selected S. mutans being designated as clinical isolate B (6). The zone of inhibition was 72 mm and lactic acid production was 0.010 g/l at 10% xylitol concentration in Brain Heart Infusion Broth.

Statistical approach to study the interactive effects of process parameters for enhanced xylitol production by Candida tropicalis and its potential for the synthesis of xylitol monoesters

Previous results showed that an indigenously isolated yeast strain of Candida tropicalis was found to produce 12.11 g/L of xylitol under unoptimized conditions in presence of 50 g/L of xylose. In the present study, optimizing the process using one-variable at-a-time resulted in the production of 59.07 g/L of xylitol in 96 h in presence of 100 g/L xylose. Further optimization using response surface methodology led to the production of 65.45 g/L in medium containing 100 g/L xylose, 0.5% yeast extract, 0.03% MgSO4.7H2O and 0.2% KH2PO4, pH-4.5, 30 °C, 200 r/min for 96 h with 4% inoculum level. Addition of 1% methanol in response surface methodology optimized–medium led to the production of 67.12 g/L. Scaling up in 10 L fermentor resulted in productivity of 0.80 g/Lh with yield of 0.68 g/g. Efficient synthesis of xylitol esters was achieved with butyric acid (50.32%) and caproic acid (38.36%) in 4 h using Pseudomonas aeruginosa lipase in t-butanol: tetrahydrofuran (1:1 v/v).

Probiotics: Nutritional Therapeutic Tool

The advent of various health care policies and digital revolution has fuelled interest in the direction of food supplements, and as preventive or curative drug containing live non-pathogenic bacteria, probiotics. The widespread usage of probiotics by consumers and in clinical practice has emphatically made to understand the benefits associated with these products. The probiotic products are now being marketed worldwide by several multinational companies. Present review attempts to overview the health benefits of probiotics in impeding various lifestyle or metabolic associated disorders, clinical significance, their efficacy and influence on immune system.

Efficient 1–5 regioselective acylation of primary hydroxyl groups of fermentative derived xylitol catalyzed by an immobilized Pseudomonas aeruginosa lipase

The experiment described in this paper synthesizes xylitol acylated products from fermentative derived xylitol and acid anhydrides of various chain lengths in the presence of Tetrahydrofuran (THF) and acetonitrile using immobilized Pseudomonas aeruginosa (PL) lipase as a biocatalyst (97% residual activity up to five cycles) at 37°C, 200 rpm. This study examines a number of different acid anhydrides for their highly selective and efficient lipase-catalyzed acylation of primary hydroxyl groups in xylitol. Of those studied, the best results are obtained with butanoic anhydride, 80.12% after 4 h in acetonitrile followed by vinyl acetate, which results in 77.79% conversion after 8 h of incubation in THF as analyzed through high performance liquid chromatography (HPLC).