11 Seaweed sample was collected by hand picking at a depth of 1–2 m in Gulf of Mannar, Southeast Coast of India. The samples were surface sterilized with natural seawater followed by double distilled water in the laboratory. The seaweed samples were identified as S. tenerrimum. Seaweed material as a whole was shade dried for 15 days to prevent photolysis and powdered with a mixer grinder. The solid liquid extraction (Soxhlet extraction) was performed with dried seaweed powder of 25 g in 200 ml of methanol (purity grade 99%). The extraction was done for
about 12 h at 35 °C until the colour of the seaweed turns from dark brown to pale brown. AUY922 Later, the soxhleted material was removed and concentrated under reduced pressure to as low as 1 ml using a rotary evaporator (Buchi, Switzerland) and refrigerated at −4 °C. FT-IR analysis was performed with a mixture containing powdered potassium bromide (KBr) and lyophilized methanolic seaweed extract. The molecular functional vibrations of chemical groups present in the sample was recorded with Perkin-Elmer FT-IR spectrum – 1 spectrophotometer operated at a resolution of 2 cm−1 ranging from 4000 to 400 cm−1. The Gas Chromatography–Mass Spectrometry (GC–MS) analysis was performed with a GC–MS (Shimadzu QP-2010 Plus – Tokyo, Japan)
of thermal Desportion System TD 20. The system was equipped with HP-5MS capillary column of 30 m × 0.25 mm and 0.25 μm of film thickness. The ionization energy used in the present Metalloexopeptidase study was about 70 eV. Helium gas (99.999% purity) was find more used as a carrier gas at a constant flow rate of 1.21 ml/min. One μl of samples was injected in the split mode with 10:0 ratios.
The GC injector and MS transfer line temperatures were set at 230 and 280 °C respectively. The ion source temperature was constantly maintained at 300 °C. Oven temperature programme was initially set at 100 °C with a hold time of 2 min. Further, it was ramped to 200 °C (at 5 °C/min) with the hold time of 5 min and to 235 °C (at 10 °C/min) with the hold time of 10 min. The resulting peaks were analyzed in inbuilt mass spectrum library such as NIST05.LIB and WILEY8.LIB. Antibacterial activity of methanolic extracts was evaluated by disk diffusion technique. Pathogenic bacterial strains such as Escherichia coli (MTCC 1687), Klebsiella pneumoniae (MTCC 530), Pseudomonas aeruginosa (MTCC 1688), Salmonella typhii (MTCC 531), Staphylococcus aureus (MTCC 96) and Vibrio cholerae (MTCC 3906) were procured from Microbial Type Culture Collection (MTCC), Indian Institute of Microbial Technology, Chandigarh, India. The pathogens were inoculated in Luria Bertani (LB) broth and kept overnight at 37 °C for exponential growth of cultures. Later, the bacterial cultures (106 CFU ml−1) were swabbed on freshly prepared LB plates and sterile disks of 6 mm (HIMEDIA) were placed on the plate.