A master thesis submitted in partial fulfillment of the requirements for the Degree of Master of Science in Food Engineering and Bioprocess Technology, School of Environment, Resources and Development

Thesis (M. Sc.) - Asian Institute of Technology, 2017

The emergence of antibiotics resistance in food -borne pathogens is due to excessive use of antibiotics for therapeutic and prophylactic purposes. The management of multidrug resistant bacteria with available antibiotics is becoming more difficult task and is of great food safety concern. The test organisms ( Escherichia coli, Staphylococcus aureus, Salmonella typhi and Listeria monocytogenes ) used in current study was found resistant to ampicillin (10 og), tetracycline (30 og), sulfamethoxazole (25 og) and amoxicillin (10 og). Among all test organisms Escherichia coli was also found resistant to fourth generation antibiotics, i.e. norfloxacins. Further objective of current study was to identify bioactive compounds from Acacia nilotica extracts and Lactobacillus plantarum for synergistic antibacterial, antimutagenic and cytotoxic effects. The study was conducted to optimize the ultrasound assisted extraction (UAE) of bioactive compounds from Acacia nilotica leaves using Design Expert 10.0.0. The effects of three independ ent variables (solvent concentrations, solvent volumes and extraction times) on total phenolic content ( TPC ), total flavonoids content ( TFC ), percentage 2.2- diphenyl -2- picrylhydrayl hydrate ( DPPH ) inhibition, ferric reducing antioxidant power ( FRAP ), reducing power and antibacterial activity was examined by response surface methodology (RSM). The optimized conditions for extraction of bioactive compounds were 58.6% ethanol (v/v), 10 ml volume of solvent per g of raw materials for 20.6 min extraction time. Under these conditions, TPC, TFC, DP P H inhibition, FRAP values and reducing power were 979.90 mg gallic acid equiva lent/ g, 41.1 mg quercetin equivalent/ g , 79.7%, 10619.3o M/g and 849.3 mg ascorbic acid acid equivalent / g respectively. Statistically, only solvent concent ration has significant (p - value< 0.05) effect on the bioactive properties of extracts. All the ethanolic extracts were found having antibacterial activity against multidrug resistant bacteria ( E. coli, S. aureus, S. typhi and L. monocytogenes ). The ethanolic extract showed higher TPC , TFC , antioxidant activity and antibacterial activity than aqueous extracts. The presence of some s imila r functional groups of active compounds was confirmed in partially purified and crude extract of Acacia nilotica leaves by Fourier transform infrared spectroscopy (FTIR). Further, synergistic antibacterial effects between Acacia nilotica extract and CFS from L. plantarum was evaluated to reduce the required minimum inhibitory concentration ( MIC ) and increases its industrial applications. The Lactobacillus plantarum produces high amount of antibacterial compounds within 60 h of incubation at 37{u00BA} C. The 20 X CFS shows antibacterial activity against all test organisms. The minimum inhibitory concentration of Acacia nilotica extract and CFS were 6.25 mg/ml and 50 ol/ml respectively. The synergy between Acacia nilotica extract and CFS was obtained at combination (1/2MIC extract X 1/4CFS and 1/4MIC extract X 1/2CFS) which correspond to FIC index 0.75. The ext ract and CFS alone and its combination show strong antimutagenicity against Salmonella typhimurium TA 100 and inhibit the growth of Salmonella on chicken meat for 3 days of storage at 8 {u00BA}C. The Acacia nilotica (500 og/ml ) show cyto toxicity on Caco 2 cell line with IC 50 value 397.10. The synergy combination of Acacia nilotica with C FS show C ytotoxicity at 2 % and 1 % concentration with IC 50 value 0.915. Whereas CFS alone shown non -cyto toxic effect on Caco2 cell line. This study shows that synergistic combination between Acacia nilotica extract and Lactobacillus plantarum CFS can be effective approach in bio-preservation of perishable food and alternatives to manage emergence of antibiotics resistant pathogens