Effect of Eleutherine americana Merr. Bulb Extracts on Food Poisoning Staphylococcus aureus and its Application in Food Systems Ifesan Beatrice Olawumi Temilade A Thesis Submitted in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Microbiology Prince of Songkla University 2009 Copyright of Prince of Songkla University i Thesis Title Effect of Eleutherine americana Merr. Extracts on Food Poisoning Staphylococcus aureus and Its Application in Food Systems Author Mrs. Ifesan Beatrice Olawumi Temilade Major Program Microbiology Major Advisor: Examining Committee: ……………………………………… ……………………………Chairperson (Assoc. Prof. Dr. Supayang Voravuthikunchai) (Assoc. Prof. Dr. Nongyao Sawangjaroen) ………………………………………… Co-advisor: (Assoc. Prof. Dr. Supayang Voravuthikunchai) ……………………………………… ………………………………………… (Dr. Sunisa Siripongvutikorn) (Asst. Prof. Dr. Pongsri Tongtawe) ………………………………………... (Dr. Sunisa Siripongvutikorn) The Graduate School, Prince of Songkla University, has approved this thesis as fulfillment of the requirements for the Doctor of Philosophy Degree in Microbiology ………………………………………... (Assoc. Prof. Dr. Krerkchai Thongnoo) Dean of Graduate School ii Thesis Title Effect of Eleutherine americana Merr. Extracts on Food Poisoning Staphylococcus aureus and Its Application in Food Systems Author Mrs. Ifesan Beatrice Olawumi Temilade Major Program Microbiology Academic Year 2009 Abstract Bulbs of Eleutherine americana Merr. were examined for their antibacterial activities against Staphylococcus aureus isolated from foods. Ready-to- eat foods were purchased over a period of 3 months out of which 76 (71.69%) were contaminated with Staphylococcus aureus. The isolates were characterized phenotypically using traditional biochemical methods. Ninety-four percent of the isolates were mannitol fermenters, 86% positive for coagulase test, while 80% produced lipase enzyme. Antibiotic susceptibility test revealed that 21% and 63% of the food isolates were resistant to oxacillin and penicillin, respectively. The results showed that 22 (20.75%) food samples were contaminated with methicillin-resistant S. aureus (MRSA). The antibacterial activity of the acetone, ethanol, ethanol+hexane, and hexane extracts from the bulbs of E. americana were investigated by paper disc agar containing 2.5 mg of the crude extract. The various solvents produced similar inhibition zones, ranging from 14.5 and 15.7 mm for the food isolates, and 12.2-17.0 mm for the enterotoxin-producing reference strains. The minimal inhibitory concentration (MIC) values ranged from 0.06 to 1.00 mg/ml for food isolates and 0.25 mg/ml for the three reference strains. Growth curve in the presence of the crude ethanol extract at 4MIC showed bacteriostatic effect by 5 log reduction relative to the control. The ethanolic extract from the bulb of E. americana was investigated for its ability to inhibit enzymes and enterotoxin production by S. aureus. Preliminary iii screening of the isolates for enzyme synthesis revealed that higher percentage of the isolates produced lipase more than protease enzymes. About 15% of the 106 isolates were positive for enterotoxin production with staphylococcal enterotoxin A (11.3%), enterotoxin B (3.7%), enterotoxin C (10.3%), and no enterotoxin D was produced. The production of staphylococcal enterotoxins A–D in the presence or absence of the crude extract was carried out. In the broth system, the extract reduced enterotoxin production at subminimal inhibitory concentrations compared with the control. At MIC, total enterotoxin inhibition was observed for enterotoxin C production, whereas synthesis of enterotoxins A, B, and D were totally eliminated at 2MIC. The food system study revealed that the extract could delay production of enterotoxins A, B, and C compared with the control. The extract at 2 mg/ml delayed production of toxins A and C for 8 and 4 h, while toxin B was not detected in the pork at 48 h. The ability of E. americana extract to inhibit lipase and protease enzymes and to delay enterotoxin production in food could present it as a novel food additive to combat the growth of S. aureus in food. The mechanisms of action of ethanolic extract from E. americana against S. aureus was investigated. Treatment of S. aureus ATCC 27664 with crude extract at 2MIC reduced the inoculum size by 5 log at 24 h compared with the control. The combined effect of the extract and 7.5% NaCl on the enterotoxin-producing ATCC strain resulted to total elimination within 24 h, compared to the control. The release of cell materials after extract treatment was determined by measuring optical density of the suspensions at 260 nm. It was observed that the treatment resulted in cytoplasmic leakage. Optical density determination at 620 nm showed that the extract did not cause gross cell wall damage. However, observation of staphylococcal cells treated with 2MIC and 4MIC of the crude extract under electron microscope revealed that E. americana caused damages to membrane integrity with some dark spots in the cytoplasm. A knowledge and understanding of the mechanism of action of E. americana extract could offer useful hints in the search for novel antibacterial both in clinical and food system. The ethanolic extract was investigated for its antistaphylococcal activity both in vitro and in different food systems. The extract activity against S. iv aureus was better at 35ºC than at 10ºC and 4ºC, respectively. The extract exhibited excellent stability to heat and pH treatments. The scavenging activities of crude ethanolic extract from E. americana was investigated. The results revealed that the extract produced IC50 values of 8.4 µg/ml and 0.78mg/ml on 2,2-diphenyl-1- picrylhydrazyl and hydroxyl free radicals, respectively. Total phenolic content of the extract was determined using the Folin–Ciocalteu reagent and the crude extract yielded high phenolic content of 4.56 µmol gallic acid equivalent/mg dried extract. The crude extract was incorporated into home made salad dressing and examined for its antibacterial, physical, chemical, and sensory properties during storage at 4ºC for 16 days. A reduction of more than one log in S. aureus count was observed, compared to the control. Similarly, the extract at different concentrations were incorporated into pork, cooked in the microwave, and stored at 4ºC for 9 days. Antibacterial activity of the extract against S. aureus in the cooked pork revealed that addition of 10.8 mg/100 g of the extract reduced the bacterial population by 0.57 log compared to control at 9 day of storage. The crude extract demonstrated antioxidant activity which increases with increased extract concentration and retarded lipid oxidation in the salad dressing and cooked pork. Furthermore, addition of the extract led to increase in the redness values of the pork and this was acceptable from the sensory point of view. The sensory evaluations for both salad dressing and cooked pork revealed that the products at all extract concentrations were acceptable. The extract from E. americana can be a promising novel additive to improve the quality and safety of home made salad dressing, as an antioxidant to prevent lipid oxidation and a potential natural colour enhancer of red meat and meat products. Antimicrobial activity of the crude ethanolic extract was further examined using fourteen strains of bacteria, six fungi, and two yeasts. The extract demonstrated good antibacterial activities and produced inhibition zones ranging from 13.0-20.0 mm against all the Gram-positive bacteria tested, and only one out of the Gram-negative bacteria. In addition, the extract exhibited antifungal activity against Aspergillus niger, Rhizopus spp., and Penicillium spp. while all the dermatophytic fungi and yeast strains were resistant to the extract treatment. Growth curve in the presence of the crude ethanol extract at 4MIC showed bacteriostatic and fungistatic v effects by 5 and 3 log reduction, respectively relative to the control. Data from this study revealed that E. americana bulb may be a good antimicrobial agent against foodborne pathogen and food spoilage organisms. Partially-purified fractions from E. americana extract were identified by column chromatography. The fractions were examined for their antibacterial activity on MRSA isolates obtained from foods. Fraction Ea6.3 produced MIC values of 125-500 µg/ml and minimal bactericidal concentration (MBC) of 250->1,000 µg/ml, whereas fraction Ea9 yielded MIC/MBC of 125-250/500->1,000 µg/ml against all the MRSA isolates. The MIC/MBC values for the enterotoxin-producing reference strains were 250/500 µg/ml for Ea6.3 while Ea9 produced MIC/MBC values of 125/>1,000 µg/ml. Growth curves in the presence of fraction Ea6.3 at 4MIC resulted in total elimination of all the test strains between 20 and 24 h, while fraction Ea9 reduced bacterial population by at least 6 log relative to the control. The partially- purified fractions were further purified to obtain pure compounds which produced MICs ranging from 31.25 to 1,000 µg/ml against S. aureus reference strains. vi ACKNOWLEDGEMENT I dedicate this thesis to the Almighty God, Jehova Sabaoth, who has been my help, support, strength, and granted me the opportunity to attain this height in my career. He makes all things beautiful in His own time. I am equally grateful to the Graduate School, Prince of Songkla University for the scholarship given to me to undertake this study. I appreciate the support, love and motherly role played by my major supervisor, Assoc. Prof. Dr. Supayang Piyawan Voravuthikunchai to make this research a success. I am grateful to Dr. Sunisa Siripongvutikorn for her contributions to this work. I am equally grateful to Prof. Darah Ibrahim for the opportunity given to me to carry out research in her laboratory in Universiti Sains Malaysia. I also appreciate my thesis examination committee Asst. Prof. Dr. Pongsri Tongtawe, and Assoc. Prof. Dr. Nongyao Sawangjaroen. I am grateful to all the staff of Microbiology Department, Prince of Songkla University. I appreciate all my friends in Prince of Songkla University, especially members of PR 534 for their love and contributions to my success.