Cow Dung Is a Novel Feedstock for Fibrinolytic Enzyme Production from Newly Isolated Bacillus sp. IND7 and Its Application in In Vitro Clot Lysis

Bacterial fibrinolytic enzymes find great applications to treat and prevent cardiovascular diseases. The novel fibrinolytic enzymes from food grade organisms are useful for thrombolytic therapy. This study reports fibrinolytic enzyme production by Bacillus sp. IND7 in solid-state fermentation (SSF). In this study, cow dung was used as the cheap substrate for the production of fibrinolytic enzyme. Enzyme production was primarily improved by optimizing the nutrient and physical factors by one-variable-at-a-time approach. A statistical method (two-level full factorial design) was applied to investigate the significant variables. Of the different variables, pH, starch, and beef extract significantly influenced on the production of fibrinolytic enzyme (p < 0.05). The optimum levels of these significant factors were further investigated using response surface methodology. The optimum conditions for enhanced fibrinolytic enzyme production were 1.23% (w/w) starch and 0.3 % (w/w) beef extract with initial medium pH 9.0. Under the optimized conditions, cow dung substrate yielded 8,345 U/g substrate, and an overall 2.5-fold improvement in fibrinolytic enzyme production was achieved due to its optimization. This is the first report of fibrinolytic enzyme production using cow dung substrate from Bacillus sp. in SSF. The crude enzyme displayed potent activity on zymography and digested goat blood clot completely in in vitro condition

Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60\ub0C and was stable up to 50\ub0C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent

Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues by Bacillus cereus IND1 and Its Thrombolytic Activity In Vitro

A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P<0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent

Medium Optimization for the Production of Fibrinolytic Enzyme by Paenibacillus

Production of fibrinolytic enzyme by a newly isolated Paenibacillus sp. IND8 was optimized using wheat bran in solid state fermentation. A 25 full factorial design (first-order model) was applied to elucidate the key factors as moisture, pH, sucrose, yeast extract, and sodium dihydrogen phosphate. Statistical analysis of the results has shown that moisture, sucrose, and sodium dihydrogen phosphate have the most significant effects on fibrinolytic enzymes production (P<0.05). Central composite design (CCD) was used to determine the optimal concentrations of these three components and the experimental results were fitted with a second-order polynomial model at 95% level (P<0.05). Overall, 4.5-fold increase in fibrinolytic enzyme production was achieved in the optimized medium as compared with the unoptimized medium

Cow dung is an ideal fermentation medium for amylase production in solid-state fermentation by Bacillus cereus

Amylase production by Bacillus cereus IND4 was investigated by solid state fermentation (SSF) using cow dung substrate. The SSF conditions were optimized by using one-variable-at-a-time approach and two level full factorial design. Two level full factorial design demonstrated that moisture, pH, fructose, yeast extract and ammonium sulphate have significantly influenced enzyme production (p < 0.05). A central composite design was employed to investigate the optimum concentration of these variables affecting amylase production. Maximal amylase production of 464 units/ml of enzyme was observed in the presence of 100% moisture, 0.1% fructose and 0.01% ammonium sulphate. The enzyme production increased three fold compared to the original medium. The optimum pH and temperature for the activity of amylase were found to be 8.0 and 50 °C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 32% enzyme activity after initial denaturation at 50 °C for 1 h. This is the first detailed report on the production of amylase by microorganisms using cow dung as the low cost medium

Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology

Cardiovascular diseases (CVDs) cause high mortality throughout the world. Existing fibrinolytic agents are highly expensive and have many side effects. Microbial fibrinolytic enzymes are very much considered as novel therapeutic candidate for the treatment of CVDs. Reports on fibrinolytic enzyme from Xanthomonas sp. is lacking. This study reports fibrinolytic enzymes from Xanthomonas oryzae IND3 as it shows hyperactivity on fibrin-agarose plates. This organism utilized various agro-industrial wastes for enzymes production. Among all, cow dung enhanced more enzyme production, hence it was used as the low-cost substrate for statistical optimization of fibrinolytic protease in Solid state fermentation. Response surface methodology was employed to optimize the factors and enhanced yield by 4-fold. The interactions among the variables, viz, sucrose, yeast extract, and pH of the medium were investigated using Central Composite Design (CCD). The predicted fibrinolytic enzyme activity was 2340 U/g, and the observed fibrinolytic enzyme activity was 2294 ± 12.8 U/g. The fibrinolytic enzyme degraded blood clot in vitro completely. This study is the first report on statistical optimization of fibrinolytic enzyme production in SSF from Xanthomonas sp. The crude extract has immense activity on proteinaceous wastes. The production of fibrinolytic protease using the low-cost substrate could reduce the production cost of enzyme. Keywords: Cardiovascular diseases, Xanthomonas oryzae, Solid-state fermentation, Fibrinolytic enzyme, Response surface methodology, Blood clot lysi

Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60°C and was stable up to 50°C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent

Probiotic Characteristics of Ligilactobacillus salivarius AS22 Isolated from Sheep Dung and Its Application in Corn-Fox Tail Millet Silage

The forage crops corn (Zea mays) and foxtail millet (Panicum italicum L.) are widely used as animal feed because of their high nutritive values. The ensiling of corn and foxtail millet is often associated with the growth of undesirable microbes, which cause severe loss of dry matter content during the storage periods. The selection of suitable Ligilactobacillus species for corn-fox tail millet silage production can improve the quality. In this study, we aimed to select potent lactic acid bacteria (LAB) from sheep dung and analyses their biological application such as probiotic features, antimicrobial activities and fermentation capability of silage. A total of nine Lactobacillus strains were inoculated in MRS medium to evaluate lactic acid concentration. The isolated strain, Ligilactobacillus salivarius AS22, produced a higher lactic acid level (40.2 ± 2.2 µg/mL) with high growth rates (2.24 ± 0.12 OD at 600 nm) compared to other strains. The silage treated with inoculant (L. salivarius AS22) decreased the pH value (p &lt; 0.05) and enhanced lactic acid production (p &lt; 0.05) than the control at ensiled silages. LAB inoculated silage had reduced numbers of fungal colonies than control (p &lt; 0.05). In conclusion, the addition of L. salivarius AS22 improved the quality of whole corn and foxtail millet silages with significant probiotic potential