ABSTRACT
Rhamnolipid biosurfactant produced by Pseudomonas aeruginosa, possesses non-toxicity, environmental compatibility, a wide range of pH (4-8), temperature (4-100 °C), and salinity (1-10%) stability. The application of RLs is worldwide accepted in the pharmaceutical, medicinal, and food industries. It has been used for cytotoxicity efficacy analysis with a limited number of cancerous cell lines. To widen the scope of rhamnolipid application as an anticancer agent, we have studied Di-RLs homolog, 'Rha-Rha-C10-C10' produced by Pseudomonas aeruginosa RA5 against human cancerous cell lines including breast cancer (MCF-7), leukemia (K-562), cervical cancer (HeLa), Lung cancer (HOP-62), and colon cancer (HT-29) in a dose-dependent way. It was purified with silica gel chromatography followed by TLC and mass spectroscopy prior to cytotoxicity analysis. With a tensiometer, critical micelle concentration of Di-RLs was estimated to be 33.92 ± 2 mN/m at 0.2%. Cytotoxicity analysis of Di-RLs on K-562 cell line demonstrated inhibition with GI50 and TGI at < 10 µg/mL and 66.6 µg/mL, after 48 h of application. The morphology of human cancerous cell lines was observed under a laser confocal microscope with the SRB staining method. Further research is recommended to comprehend the Di-RLs as a potential anti-cancer agent.
ABSTRACT
BACKGROUND AND AIM: Little information about the stability and changes of sheep ruminal microbiota due to pathogen intervention in the rumen simulation technique (RUSITEC) is available. This study aimed to investigate the effect of administration of a novel isolated Streptococcus bovis strain on rumen microbiology and physiology. In addition, the isolation of pigment-producing Streptococcus lutetiensis is described. MATERIALS AND METHODS: Microbial strains were isolated from sheep rumen digesta. An isolated strain of S. bovis was evaluated in the RUSITEC system fed with mixed cattle feed and compared with an in-house developed probiotic formulation (PF), PF 1, containing Bacillus amyloliquifaciens, Bacillus subtilis, and Propionibacterium freudenreichii. The parameters of volatile fatty acid, lactic acid, pH profiling, and the coliform anti-pathogenicity were evaluated to determine the effect of S. bovis on rumen function and physiology. RESULTS: Administration of S. bovis reduced the coliform count by 31.20% from 7.2×1010 colony-forming units (CFU)/mLto 1.7×106 CFU/mL. Agar diffusion assays revealed the extracellular antimicrobial activity of S. bovis against coliforms; Escherichia coli and Salmonella enterica with 12 and 14 mm zones of inhibition, respectively. Simultaneously, an increase of 61.62% in the rumen yeast count was noted. The physiological changes resulted in a 5% reduction in acetic acid concentration from 431 to 405 mg/L. CONCLUSION: The present research indicates that S. bovis is highly capable of altering rumen physiology and function on colonization and is a key transition microbe to be studied during rumen intervention studies. A decrease in the coliform count could be attributed to extracellular production of a bacteriocin-like substance, as illustrated through agar diffusion assays.
ABSTRACT
BACKGROUND AND AIM: A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen. MATERIALS AND METHODS: Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality. RESULTS: Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A260nm/A280 nm) and 2.3 (A260nm/A230 nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at -80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium. CONCLUSION: To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.
