Siddalingaiya Gurudutt Prapulla

Response surface methodological approach for the synthesis of isobutyl isobutyrate

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used for the synthesis of isobutyl isobutyrate by direct esterification of isobutyric acid and isobutyl alcohol. Response surface methodology based on a five-variable central composite rotatable design was used to determine the effect of solvent (log P ranging from 0.49 to 4.5), acid concentration (0.05–0.25 M), alcohol to acid ratio (1.25:1), enzyme concentration (25–225 mg), incubation period (24–120 h) and incubation temperature (30–70°C) on the esterification reaction. The extent of esterification was good at all acid concentrations employed in the range 0.05–0.25 M with increase in enzyme concentration. Even with a low enzyme concentration of 25 mg, 50% conversion was observed. The enzyme was active at all the temperatures ranging from 30 to 70°C even up to an incubation period of 120 h. The results obtained show that the yields were higher in solvents having log P>2, i.e., when solvents of higher non-polarity were employed. Optimum conditions for predicting maximum ester yield were 200 mM using 0.2 M, isobutyric acid; 0.2 M, isobutanol; enzyme concentration, 225 mg; incubation period, 72 h and hexane (log P=3.5) as reaction media at reaction temperature, 70°C. Under the above mentioned conditions, the experimental yield was 195 mM, which is well matched with the predictive yield.

Value addition to corncob: production and characterization of xylooligosaccharides from alkali pretreated lignin-saccharide complex using Aspergillus oryzae MTCC 5154.

Comparison of various pretreatments such as mild alkali/acid treatments and pressure cooking of corncob to expose its lignin-saccharide complex has been carried out to enhance enzymatic hydrolysis of xylan to xylooligosaccharides (XOS). Scanning electron micrographs of lignin-saccharide complex of native and pretreated corncob powder showed that the complex was greatly altered during alkali pretreatment. Hydrolysis of alkali pretreated corncob powder using a commercial endoxylanase produced 81+/-1.5% of XOS in the hydrolyzate equivalent to 5.8+/-0.14 mg ml(-1) of XOS. Reaction parameters for the production of XOS from corncob using endoxylanase from Aspergillus oryzae MTCC 5154 were optimized and an XOS yield of 10.2+/-0.14 mg ml(-1) corresponding to 81+/-3.9% with 73.5% xylobiose was obtained. HPLC/RID and ESI/MS analysis of XOS mixture and purified fractions showed that XOS was a mixture of neutral oligosaccharides of DP, 2-7.

The influence of water activity on the lipase catalyzed synthesis of butyl butyrate by transesterification

The influence of water activity on the synthesis of butyl butyrate by transesterification reaction (alcoholysis) using lipases from different sources was studied. A direct relationship between water activity (aw) and reaction rate was observed. The lipases exhibited an independent water activity profile for maximum transesterification rate. Maximum esterification yield (92%) was obtained with Candida rugosa lipase when compared to the yields from other lipases (Rhizopus oryzae , 45%; Mucor javanicus , 84%; Aspergillus niger , 56% and Penicillium roqueforti , 67%). At higher water activity (aw: 0.96) the reaction equilibrium favoured the hydrolysis of ethyl butyrate and the equilibrium was shifted to synthetic mode (butyl butyrate) when operated at low water activity (0.33) levels. Optimum water activity for obtaining maximum yield varied with the concentration of substrate used for the reaction. When substrate concentrations of 50 and 100 mM were used the maximum yields were obtained at aw of 0.33. When substrate concentrations of 150 and 200 mM were used maximum yields were obtained at aw of 0.54. At higher water activity levels (aw 0.96)almost all the enzymes showed lower rates which could be due to protein aggregation.

Enzymic synthesis of isoamyl isovalerate using immobilized lipase from Rhizomucor miehei: a multivariate analysis

Isoamyl isovalerate, an ester with apple flavour was synthesized using Rhizomucor miehei lipase immobilized on a weak anion-exchange resin (Lipozyme IM-20) by the direct esterification of isoamyl alcohol and isovaleric acid. Various reaction parameters including substrate concentration, enzyme concentration, substrate molar ratio, temperature and incubation time affecting the synthesis of isoamyl isovalerate were investigated. A maximum yield of >85% was obtained at 0.5 M (acid) substrate concentration (1.5:1 alcohol/acid ratio) using 10 g/l enzyme concentration and a reaction temperature of 50°C for 144 h incubation time in n-hexane. A solvent screening study indicated that hydrophobic solvents including cyclohexane, hexane, heptane/isooctane are the most suitable ones for esterification. The feasibility of synthesizing the ester under solvent-free conditions has also been studied. However, the conversions were very low. The parameters influencing the yield were modeled using multivariate analysis. These equations can be applied to predict the yield at any other known values of the input parameter.

Production of fructo-oligosaccharides by fructosyl transferase from Aspergillus oryzae CFR 202 and Aureobasidium pullulans CFR 77

Fructo-oligosaccharides (FOS) can be produced by the action of fructosyl transferases from plants and microorganisms. The production of FOS was attempted using culture fluid, cells and culture broth homogenate from Aspergillus oryzae CFR 202 and Aureobasidium pullulans CFR 77 using different concentrations of sucrose as substrate. The maximum yield of FOS obtained using A. oryzae CFR 202 culture fluid was 50–54% after a reaction time of 12 h at 55 ◦C at sucrose concentrations of 55% (w/v) and 80% (w/v). The culture broth of A. oryzae CFR 202 gave 50–53% FOS after 12 h of reaction when 55% (w/v) sucrose was used as substrate. Although the cells were not suitable for FOS production, the culture fluid and culture broth homogenate of A. pullulans CFR 77 gave a conversion yield of up to 57% from sucrose at concentrations of 55 and 80% (w/v).

Beneficial effect of xylo-oligosaccharides and fructo-oligosaccharides in streptozotocin-induced diabetic rats

The beneficial influences of xylo-oligosaccharides (XOS) obtained from alkali-pretreated corncob and fructo-oligosaccharides (FOS) obtained from cane sugar were evaluated in experimental diabetes. These oligosaccharides were supplemented at 10 % (w/w) in the basal diet of streptozotocin-induced diabetic Wistar rats, while the control rats were fed with a basal diet for a period of 6 weeks. Both the oligosaccharides exerted favourable influences in diabetic rats by significantly improving body weight and reducing hyperglycaemia and cholesterol. The characteristic diabetic complications such as severe glucosuria, proteinuria and advanced glycation end products in renal tissue, diabetic nephropathy, and blood creatinine and urea concentrations were notably reduced. Besides, these oligosaccharide supplementations significantly increased the activity of antioxidant enzymes - catalase and glutathione reductase - in the blood of diabetic rats. Supplementation of XOS and FOS resulted in a significant increase in the bifidobacteria and lactobacilli population in the caecum. The present study indicates that XOS and FOS have an ameliorating influence on metabolic abnormalities associated with diabetes, besides conferring an optimal milieu of lactobacilli and bifidobacteria, thus suggesting their potential health benefit in diabetics.

Corncob-Induced endo-1,4-β-D-Xylanase of Aspergillus oryzae MTCC 5154 : Production and Characterization of Xylobiose from Glucuronoxylan

Eight different fungi were cultivated in a peptone-yeast extract medium containing 1% oat spelt xylan (OSX) to evaluate endo-1,4-beta-xylanase secretion for xylooligosaccharide (XOS) production. Aspergillus oryzae MTCC 5154, Aspergillus flavus , Aspergillus niger , and Aspergillus ochraceus showed significant titers of endoxylanases, which were further used for the production of XOS from birch wood xylan (BWX). A. oryzae produced 89.5 +/- 1.13% XOS in the hydrolysate at 24 h of reaction. The effect of OSX, BWX, and raw corncob on the induction of endoxylanase in A. oryzae was studied, and the xylanase activity was maximum at 96 h of cultivation in 3% corncob containing medium. XOS produced at 36 h of reaction was 5.87 +/- 0.53 mg/mL (12 +/- 2% xylose, 48 +/- 2.43% xylobiose, and 40 +/- 3.6% higher oligomers) from 1% BWX . HPLC/refractive index detection and ESI/MS analysis of fractions obtained by GPC corresponded to neutral and 4- O-methyl-alpha- d-glucuronic acid substituted acidic oligosaccharides. The major fraction, beta- d-xylopyranosyl-(1-->4)- d-xylanopyranose was characterized using (13)C NMR.

Role of Cryoprotectants on the Viability and Functional Properties of Probiotic Lactic Acid Bacteria during Freeze Drying

Effect of different cryoprotectants on acid/bile tolerance, β-galactosidase activity, and cholesterol assimilation properties of freeze dried Pediococcus acidilactici, Lactobacillus plantarum, and Lactobacillus salivarius were evaluated. The retention of probiotic properties and viability of the above three cultures freeze dried with lactose, skimmed milk, and maltodextrin were found to be significant (P ≤ 0.05). In another approach, lactic acid bacteria (LAB) were subjected to cold shock first and then freeze dried. Effect of cold shock treatment on retention of probiotic properties of freeze dried LAB cultures was also investigated. The cold shock treatment resulted in an increase of 6–8%, both in viability and probiotic properties, compared with LAB freeze dried without cold shock treatment. The survival of freeze dried L. plantarum, L. salivarius, and P. acidilactici was investigated over a period of 60 d storage at 4°C. The above three cultures freeze dried with maltodextrin exhibited 83%, 68%, and 60% retention in survival percentage, respectively, in comparison with initial values.

Probiotic attributes, antioxidant, anti-inflammatory and neuromodulatory effects of Enterococcus faecium CFR 3003: in vitro and in vivo evidence.

Accumulating evidence suggests that probiotic bacteria play a vital role in modulating various aspects integral to the health and well-being of humans. In the present study, probiotic attributes and the antioxidant, anti-inflammatory and neuromodulatory potential of Enterococcus faecium CFR 3003 were investigated by employing suitable model systems. E. faecium exhibited robust resistance to gastrointestinal stress conditions as it could withstand acid stress at pH 1.5, 2 and 3. The bacterium also survived at a bile salt concentration of 0.45 %, and better tolerance was observed towards pepsin and trypsin. E. faecium produced lactic acid as a major metabolic product, followed by butyric acid. Lyophilized cell-free supernatant (LCS) of E. faecium exhibited significant antioxidant capacity evaluated against 1,1-diphenyl-2-picryl-hydrazyl, ascorbate auto-oxidation, oxygen radical absorbance and reducing power. Interestingly, E. faecium, Lactobacillus rhamnosus GG MTCC 1408 and LCS showed a significant anti-inflammatory effect by negatively modulating TNF-α production and upregulating IL-10 levels in LPS-stimulated macrophage cell lines. In an in vivo mice model, the propensity of probiotic supplements to modulate endogenous oxidative markers and redox status in brain regions was assessed. Young mice provided with oral supplements (daily for 28 days) of E. faecium and L. rhamnosus exhibited diminished oxidative markers in the brain and enhanced activities of antioxidant enzymes with a concomitant increase in γ-aminobutyric acid and dopamine levels. Collectively, our findings clearly suggest the propensity of these bacteria to protect against tissue damage mediated through free radicals and inflammatory cytokines. Although the underlying molecular mechanisms need further studies, it is tempting to speculate that probiotics confer a neuroprotective advantage in vivo against oxidative damage-mediated neurodegenerative conditions.

Modelling on isoamyl isovalerate synthesis from Rhizomucor miehei lipasein organic media: optimization studies

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was employed in the esterification of isovaleric acid and isoamyl alcohol to synthesize isoamyl isovalerate in n-heptane. Response surface methodology (RSM) based on a five-level, five-variable central composite rotatable design (CCRD) was used to evaluate the effects of important variables: enzyme concentration (20–40% w/w of acid), acid concentration (0.2–1.0 M), incubation period (24–120 h), alcohol concentration (0.25–1.25 M) and temperature (30–70 °C) on the esterification yield of isoamyl isovalerate. Extent of conversion was found to be excellent at all acid and alcohol concentrations employed in the range of 0.2–1.25 M, even at low enzyme concentration (20% w/w). The optimum conditions arrived at are as follows: 35% (w/w) enzyme concentration, 1.0 M acid concentration, 1.25 M alcohol concentration and 120 h incubation period, at 35 °C. Under these conditions, the predicted value was 680 mM ester matched very well with an experimental value of 678 mM.