Synthesis of ethyl oleate employing synthetic hydrogel-immobilized lipase of Bacillus coagulans MTCC-6375

Ten polymeric hydrogels were chemically synthesized by varying the concentrations of copolymer (DMA) and cross-linker (MBAm) molecules. An alkaline lipase of Bacillus coagulans MTCC-6375 was immobilized onto a poly (MAc-co-DMA-cl-MBAm)-hydrogel support at pH 8.5 and temperature 55ºC in 16 h. The bound lipase possessed 7.6 U.g⁻¹ (matrix) lipase activity with a specific activity of 18 U.mg⁻¹ protein. Hydrogel bound-lipase catalyzed esterification of oleic acid and ethanol to synthesize ethyl oleate in n-nonane. Various kinetic parameters were optimized to produce ethyl oleate using immobilized lipase. The optimal parameters were bound enzyme/substrate (E/S) ratio 0.62 mg/mM, ethanol/oleic acid 100 mM:75 mM or 100 mM:100 mM, incubation time 18 h and reaction temperature 55ºC that resulted in approximately 53% conversion of reactants into ethyl oleate in n-nonane. However, addition of a molecular sieve to the reaction mixture promoted the conversion to 58% in 18 h in n-nonane, which was equivalent to 55 mM of ethyl oleate produced.

Synthesis of ethyl laurate by hydrogel immobilized lipase of Bacillus coagulans MTCC-6375

An alkaline thermo-tolerant lipase from Bacillus coagulans MTCC-6375 was purified and efficiently immobilized onto a synthetic hydrophobic poly (MAc-co-DMA-cl-MBAm)-hydrogel at pH 8.5 and temperature 55°C in 16 h. The hydrogel bound matrix possessed 7.6 IU g -1 matrix lipase activity with a specific activity of 18 IU mg -1 protein. Immobilized lipase was used to catalyze the esterification of lauric acid and ethanol to produce ethyl laurate in n-nonane. The reaction conditions that were optimized to produce ethyl laurate in n-nonane included enzyme/substrate (E/S) ratio, substrate concentration, reaction time and reaction temperature. The optimized parameters were E/S ratio of 0.5 mg mM -1, ethanol:lauric acid in ratio of 100 mM:100 mM and reaction time of 15 h at 65°C under continuous shaking (200 rpm). Optimized conditions resulted in 66% conversion of reactants into ethyl laurate in n-nonane in the presence of 300 mg molecular sieve mL -1 reaction mixture.

Thermdstable alpha amylase production by bacillus coagulans

Microbial enzymes are in great demand owing to their importance in several industries such as brewing, baking, leather, laundry detergent, dairy. starch processing and textiles besides pharmaceuticals. About 80% of the enzymes produced through fermentation and sold in the industrial scale are hydrolytic enzymes. Due to recognition of new and new applications, an intensive screening of different kinds of enzymes with novel properties, from various microorganisms, is being pursued all over the world. Bacillus sp are largely known to produce a-amylase, among the different groups of microoganisms, at industrial level. They are known to produce both saccharifying and liquefying a-amylases (Fukumoto 1963; walker and Campbell, 1967a). which are distinguishable by their mechanisms of starch degradation by the fact that the saccharifying asamylases produce an increase in reducing power about twice that of the liquefying enzyme (Fukumoto, 1963; Pazur and Okada, 1966). Under this circumstances, the present study was undertaken, with a View to utilise a fast growing B.coagu1ans isolated from soil, for production of thermostable and alkaline oz-amylase under different fermentation processes

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC30 supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC30 supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC30 supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC30-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC30, suggesting that a bi-modal lifecycle of GanedenBC30 in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.

Bacillus coagulans as a probiotic

Probiotics are live microbial feed additions that improve human or animal health. Their activities are towards improving the composition of the gastrointestinal microbiota in a manner that reduces the risk of disorder. In some cases, probiotics are also used therapeutically. Most probiotics use lactobacilli or bifidobacteria as the main constituents. These produce lactic acid as well as other anti-pathogenic attributes. Traditionally, probiotics are incorporated in dairy products (yoghurts or fermented drinks) or in lyophilised form. Because of stability and viability factors, heated products are not usually a target for probiotic use. This is because they are temperature sensitive. However, a spore-forming genus would have the ability to overcome this limitation. Here, we discuss evidence for the spore-forming Gram-positive bacterium Bacillus coagulans as a probiotic.

Bacillus coagulans GBI-30, 6086 modulates Faecalibacterium prausnitzii in older men and women

Abstract Background: Advancing age is linked to a decrease in beneficial bacteria such as Bifidobacterium spp. and reduced aspects of innate immune function. Objectives: We investigated whether daily consumption of a probiotic [Bacillus coagulans GBI-30, 6086 (BC30); GanedenBC30] could improve immune function and gut function in men and women aged 65–80 y, using a double-blind, placebo-controlled crossover design. Method: Thirty-six volunteers were recruited and randomly assigned to receive either a placebo (microcrystalline cellulose) or the probiotic BC30 (1 3 109 colony-forming units/capsule). Volunteers consumed 1 treatment capsule per day for 28 d, followed by a 21-d washout period before switching to the other treatment. Blood and fecal samples were collected at the beginning and end of each treatment period. Fecal samples were used to enumerate bacterial groups and concentrations of calprotectin. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood to assess natural killer cell activity and lipopolysaccharide (LPS)-stimulated cytokine production. C-reactive protein concentrations were measured in plasma. Results: Consumption of BC30 significantly increased populations of Faecalibacterium prausnitzii by 0.1 log10 cells/mL more than during consumption of the placebo (P = 0.03), whereas populations of Bacillus spp. increased significantly by 0.5 log10 cells/mL from baseline in volunteers who consumed BC30 (P = 0.007). LPS-stimulated PBMCs showed a 0.2 ng/mL increase in the anti-inflammatory cytokine IL-10 28 d after consumption of BC30 (P < 0.05), whereas the placebo did not affect IL-10, and no overall difference was found in the effect of the treatments. Conclusions: Daily consumption of BC30 by adults aged 65–80 y can increase beneficial groups of bacteria in the human gut and potentially increase production of anti-inflammatory cytokines. This study shows the potential benefits of a probiotic to improve dysbiosis via modulation of the microbiota in older persons. J Nutr doi: 10.3945/jn.114.199802.

Effect of prebiotics on the fecal microbiota of elderly volunteers after dietary supplementation of Bacillus coagulans GBI-30, 6086

In advancing age, gut populations of beneficial microbes, notably Bifidobacterium spp., show a marked decline. This contributes to an environment less capable of maintaining homoeostasis. This in vitro investigation studied the possible synergistic effects of probiotic supplementation in modulating the gut microbiota enabling prebiotic therapy to in elderly persons. Single stage batch culture anaerobic fermenters were used and inoculated with fecal microbiota obtained from volunteers after taking a 28 day treatment of Bacillus coagulans GBI-30, 6086 (GanedenBC30 (BC30)) or a placebo. The response to prebiotic supplements fructooligosaccharides (FOS) and galactooligosaccharides (GOS) in the fermenters was assessed. Bacterial enumeration was carried out using fluorescent in situ hybridisation and organic acids measured by gas chromatography. Baseline populations of Faecalibacterium prausnitzii, Clostridium lituseburense and Bacillus spp. were significantly higher in those having consumed BC30 compared to the placebo. Both prebiotics increased populations of several purportedly beneficial bacterial groups in both sets of volunteers. Samples from volunteers having ingested the BC30 also increased populations of C. lituseburense, Eubacterium rectale and F. prausnitzii more so than in persons who had consumed the placebo, this also resulted in significantly higher concentrations of butyrate, acetate and propionate. This shows that consumption of BC30 and subsequent use of prebiotics resulted in elevated populations of beneficial genres of bacteria as well as organic acid production

Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372

A purified alkaline thermo-tolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a Poly (MAc- co -DMA- cl -MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification of myristic acid and isopropanol in n -heptane at 65 °C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in n -heptane resulted in formation of isopropyl myristate (66.0 ± 0.3 mM) in 15 h. The reaction temperature below or higher than 65°C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 Å × 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 ± 0.2 mM isopropyl myristate after the 3 ^rd cycle of esterification.

Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg ²⁺ , Cu ²⁺ , Ca ²⁺ , Hg ²⁺ , Al ³⁺ and Fe ³⁺ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg ²⁺ ions synergized and Zn ²⁺ and Co ²⁺ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p -nitrophenyl palmitate and showed a V _max and K _m of 0.70 mmol.mg ⁻¹ .min ⁻¹ and 32 mM for hydrolysis of p NPP.

Synthesis of ethyl acetate employing celite-immobilized lipase of Bacillus cereus MTCC 8372

A wide range of fatty acid esters can be synthesized by esterification and transesterification reactions catalyzed by lipases in non-aqueous systems. In the present study, immobilization of a purified alkaline extra-cellular lipase of Bacillus cereus MTCC 8372 by adsorption on diatomaceous earth (celite) for synthesis of ethyl acetate via transesterification route was investigated. B. cereus lipase was deposited on celite (77% protein binding efficiency) by direct binding from aqueous solution. Immobilized lipase was used to synthesis of ethyl acetate from vinyl acetate and ethanol in n -nonane. Various reaction conditions, such as biocatalyst concentration, substrates concentration, choices of solvents ( n -alkanes), incubation time, temperature, molecular sieves (3Å × 1.5 mm), and water activity(a _w ), were optimized. The immobilized lipase (25 mg/ml) was used to perform transesterification in n -alkane(s) that resulted in approximately 73.7 mM of ethyl acetate at 55 °C in n -nonane under shaking (160 rpm) after 15 h, when vinyl acetate and ethanol were used in a equimolar ratio (100 mM each). Addition of molecular sieves (3Å × 1.5 mm) as well as effect of water activity of saturated salt solutions (KI, KCl and KNO ₃ ) to the transesterification efficiency has inhibitory effect. Batch operational stability tests indicated that immobilized lipase had retained 50% of its original catalytic activity after four consecutive batches of 15 h each.