Isolation, characterization, and cloning of thermostable pullulanase from Geobacillus stearothermophilus ADM-11.

This study aimed to identify thermo-stable pullulanase-producing bacteria in soil samples of potato fields and food-producing companies. Pullulan agar medium was used to screen 17 bacterial strains, which were incubated at 65 °C. The isolate with the maximum activity (375U/ml) was selected and recognized as Geobacillus stearothermophilus ADM-11 by morphological, biochemical characterization, and 16S rRNA gene sequencing. The pullulanase production required optimum pH of 7 and temperature of 75 °C, respectively. The electrophoresis of purified pullulanase on SDS-polyacrylamide gel revealed 83 kDa of a molecular weight that is active at 70 °C and pH 7.0. It was also stable at 90 °C but its activity was decreased by 10 % at 100 °C. The action of pullulanase was increased and stabilized by Ca+2 among the metal ions. Beta and gamma-cyclodextrins inhibited enzyme activity while ethylenediaminetetraacetate (EDTA) and phenylmethylsulfonyl fluoride (PMSF) have no significant effect on pullulanase activity. A full-length pullulanase gene was amplified from G. stearothermophilus ADM-11 using genomic DNA 2.1 kb of PCR product which was then purified and ligated in the cloning vector pTZ57R using the TA cloning technique. Colony PCR confirmed cloning on the positive clones after the pullulanase gene had been ligated and subjected to restriction digestion. It revealed 74 % similarity with the reported pullulanase gene from Geobacillus sp. 44C. The thermostability of pullulanase and its ability to degrade raw pullulan may therefore have wide-scale applications in starch processing, the detergent business, and new biotechnological applications.

Effect of probiotics on hematological parameters of male and female Wistar rats.

In the present study, the effect of probiotics on the hematology of Wistar rats was examined. Locally isolated Lactobacillus plantarum MZ707748 (Pro 1), L. plantarum MZ710117 (Pro 2), Weisella confusa MZ727611 (Pro 3), and L. plantarum MZ735961 (Pro 4) were used. One strain of probiotic, L. acidophilus-14 (Pro 5), was purchased commercially. Different groups were designed as G1, G2, G3, G4, and 5, G5/PC consisting only pro 5 and NC & 0 day were untreated. Different groups have different probiotics like G1 containing Pro 1 and Pro 2, G2 comprising Pro 3 and Pro 4, G3 containing Pro 2, Pro 3 and Pro 5, G4 having Pro 1-5, and G5 containing Pro 5. A complete count of blood, serum chemistry, fecal analysis, and histopathological examination of the thymus and liver were done. Statistical differences were seen in the complete blood count parameters (p < 0.05). No difference was observed in AST, ALT, bilirubin, albumin, IL-6, and IgA (p > 0.05) except for TP, creatinine, and globulin (p < 0.05). Fecal strains of probiotic groups were antibiotic-resistant. In males, Lactobacillus helveticus OQ152020, Enterococcus lactis OQ1519891, E. faecium OQ152017, L. gasseri OQ152017, and E. lactis OQ152019 were isolated from positive control, G1, G2, G3, and G4 respectively. In females, Enterococcus sp. OP800231, Limosilactobacillus fermentum OQ151985, E. lactis OP800267, L. plantarum OP800244, and E. faecium OQ151988 were isolated from positive control, G1, G2, G3 and G4, respectively. It was concluded that all probiotic strains were safe to use and had beneficial effects on the hematology of Wistar rats.

Evaluation of isolated probiotics on the efficacy of immune system in male and female Wistar rats.

Probiotics were isolated from fruits and vegetables. Microscopic, biochemical, and molecular tests were carried out for the characterization of strains of probiotics. To assess the effects of isolated probiotics on immunity, male and female (15 + 15) Wistar rats (n = 3) were randomly distributed into 5 groups: 0-day, negative control, positive control (commercially available Lactobacillus acidophilus-14), laboratory isolated probiotics with accession numbers; Lactobacillus plantarum (MZ707748) and Lactobacillus plantarum (MZ729681), respectively. After hematological investigations, the amounts of IgA and IgG in male and female groups were significantly different (p < 0.05). At the same time, the values of Alanine-transaminase (ALT) and Aspartate-aminotransferase (AST) in both genders were average, and there were no differences (p > 0.05). Male probiotic-treated groups had decreased levels of interleukin-6, bilirubin, and creatinine, but female probiotic-treated groups had a slight rise in bilirubin and creatinine values (p = 0.05). Cellular blood count levels of Hematocrit (HCT) and white blood cells (WBC) in male groups showed considerable differences (p < 0.05), while there were no differences (p > 0.05) in female groups. Levels of Red blood cells (RBC) and mean corpuscular hemoglobin concentration (MCHC) showed distinct changes (p < 0.05) in female groups, while these values were insignificant changes (p > 0.05) among male groups. There were considerable differences between the control and groups that were given probiotics. Histopathological results showed no damage to the liver and thymus. A fecal examination of rats was used to examine the viability and survival of Lactobacilli. Based on blood tests, it was observed that the immune system was boosted and improved in probiotic-treated groups compared to control groups.

Biotoxicity comparison of Bacillus thuringiensis to control vector borne diseases against mosquito fauna.

The current study was designed to evaluate the biotoxicity of screened echo-friendly Bacillus thuringiensis strains from different areas of Pakistan. Out of 50 samples, 36% Bt. isolates were quarantined from soil containing cattle waste after morphological, biochemical, and molecular characterization. The toxicity bioassays with Bt. spores and protein diet proved that 11 Bt. isolates were utmost noxious to 3rd instar larvae of mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The entopathogenic activity of first 4 Bt. toxins against A. aegypti was highly lethal as compared to the other dipteran larvae. The toxicity (LC50) of spore diet of Bt. strains GCU-DAB-NF4 (442.730 ± 0.38 µg/ml), NF6 (460.845 ± 0.29 µg/ml), NF3 (470.129 ± 0.28 µg/ml), and NF7 (493.637 ± 0.70 µg/ml) was quite high against A. aegypti as compared to the C. pipiens after 24 h of incubation. The highest toxicity of total cell protein was shown by GCU-DAB-NF4 (LC50 = 84.10 ± 50 µg/ml), NF6 (95.122 ± 0.40 µg/ml), NF3 (100.715 ± 06 µg/ml), and NF5 (103.40 ± 07 µg/ml) against A. aegypti after 24 h. So, these strains a have great potential to be used as biological control especially against A. aegypti as compared to the C. pipiens.