Haloalkaline Lipase from Bacillus flexus PU2 Efficiently Inhibits Biofilm Formation of Aquatic Pathogen Vibrio parahaemolyticus.

A gene encoding lipase enzyme from Bacillus flexus PU2 was cloned and expressed in E. coli BL21 (DE3) pLysS and purified protein having the molecular weight of 34 kDa. This lipase was found to be alkaline (pH 9) and slightly thermophilic. This lipase was observed to retain its activity in the presence of methanol, ethanol, DMSO, and acetone. Ferrous sulfate, copper sulfate, and manganese sulfate highly enhanced the lipase activity. All the surfactants and detergents were found to inhibit the enzyme activity, whereas the bleaching agent hydrogen peroxide was found to increase the activity. This lipase was observed as a metalloenzyme, and its activity was highly inhibited by EDTA. Also, it is moderately halophilic and can retain the activity between 0.2 and 0.8 M NaCl. Biofilm inhibitory potential of purified lipase was tested against pathogenic Vibrio parahaemolyticus, and the minimal inhibitory concentration observed was 350 U. Different concentration of this enzyme significantly changed the morphology and biofilm density of V. parahaemolyticus and was evinced by SEM and CLSM imaging. The transcriptome levels of genes responsible for biofilm formation, motility, and virulence such as, motX, fliG, and trh were significantly downregulated with lipase treatment.

Isolation, Identification of Lactobacillus mucosae AN1 and its Antilisterial Peptide Purification and Characterization.

Lactobacillus mucosae strain AN1 isolated from sheep milk and characterized for its probiotic suitability. In vitro evaluation of critical gut endurance properties of this strain were assessed by different screening methods such as bile salt, gastric acid, lysozyme tolerance assays, hemolytic, cholesterol reduction properties, and HT-29 cell line adhesion assay. Antibacterial peptide from this strain was purified using ammonium sulphate precipitation, gel filtration chromatography and reverse-phase HPLC. The molecular mass of peptides was determined by Tricine-SDS-PAGE and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF-MS). Purified peptide was named as AN1 having a molecular mass of 10.66 kDa. Helical structures of peptide were determined using circular dichroism spectroscopy. Stability of peptide AN1 towards different parameters such as pH, temperature, organic solvents, proteolytic, and glycolytic enzymes was also analyzed.

Partial purification and characterization of bacteriocin produced by Enterococcus faecalis DU10 and its probiotic attributes.

A novel bacteriocin produced by avian duck isolated lactic acid bacterium Enterococcus faecalis DU10 was isolated. This bacteriocin showed a broad spectrum of antibacterial activity against important food-borne pathogens and was purified by size exclusion chromatography followed by reverse-phase high-performance liquid chromatography in a C-18 column. Tricine-SDS PAGE revealed the presence of a band with an estimated molecular mass of 6.3 kDa. The zymogram clearly linked the antimicrobial activity with this band. This result was further confirmed by mass-assisted laser desorption ionization time-of-flight mass spectrometry, since a sharp peak corresponding to 6.313 kDa was detected and the functional groups were revealed by Fourier transform infrared spectroscopy. Bacteriocin DU10 activity was found sensitive to proteinase-K and pepsin and partially affected by trypsin and α-chymotrypsin. The activity of bacteriocin DU10 was partially resistant to heat treatments ranging from 30 to 90°C for 30 min. It also withstood a treatment at 121°C for 10 min. Cytotoxicity of bacteriocin DU10 by methyl-thiazolyl-diphenyl-tetrazolium bromide assay showed that the viability of HT-29 and HeLa cells decreased 60 ± 0.7% and 43 ± 4.8%, respectively, in the presence of 3,200 AU/mL of bacteriocin. The strain withstood 0.3% w/v of bile oxgall and pH 2 affected the bacterial growth between 2 and 4 hr of incubation. Adhesion properties examined with HT-29 cell line showed 69.85% initial population of strain E. faecalis DU10, which was found to be strongly adhered to this cell line. These results conclude bacteriocin DU10 may be used as a potential biopreservative and E. faecalis DU10 may be used as a potential probiont to control Salmonella infections.