Antibacterial Activity Of Methanobactin From Methylosinus Trichosporium 3011

In this study, methanobactin (Mb) that was obtained from fermentation of Methylosinus trichosporium, by extraction, separation, purification using a Diaion HP-20 macroporous resin column. Methanobactin (Mb) is a novel extracellular fluorescent chromopeptide produced by Methylosinus trichosporium, a methane-oxidizing bacterium. This easily culturable organism is important in global carbon cycling and for single-cell protein production. Mb has placed in a new class of compounds now known as chalkophores, which are analogous to iron-binding siderophores. Mb can be specific bind to copper and tolerance to high copper concentration. This compound fulfills multiple physiological roles including copper sequestration and/or detoxification, as well as delivery. Our research focused on the use of copper-bound methanobactin (Mb-Cu) that was studies at antibacterial activity and antibacterial mechanism, and use Mb compounded with chemical preservatives sodium benzoate and potassium sorbate. This compounds not only obtion better antibacterial effect with less dosage of preservations, but it also could moderate the conditions of bacteriostasis of food. The results offered a theoretical base for the development and utilization of bacteriocin as a kind of natural food preservative of antibacterial bacteriocin. It is believed that the application of bacteriocin of Mb as a kind of natural food preservative will be very broadly in the future.The first section of this thesis mainly discussed antibacterial activity of Mb against spoilage microorganisms. The results showed that Mb had obvious action in antibacterial upon Staphylococcus albus and Bacillus cereus of Gram-positive bacteria, and not effect in Escherichia coli of Gram-negative bacteria; But it has antibacterial activity against Escherichia coli, Staphylococcus albus and Bacillus cereus when the use of copper-bound methanobactin. To Escherichia coli, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Mb-Cu were 10.15mg/mL; To Staphylococcus albus, the MIC and MBC of Mb-Cu were 2.54 mg/mL and 5.08mg/mL; To Bacillus cereus, the MIC and MBC of Mb-Cu were 5.08mg/mL and 10.15mg/mL.In this experiment, the effects of temperature, pH value, metal ion and ultraviolet rays on Mb-Cu antibacterial activity were investigated. The results showed that the Mb-Cu had a good endurance to temperature and the antibacterial activity almost uneffected under the temperature, so it can be used in food products that require high temperature sterilization without loss of activity; Mb-Cu was active in the range of pH 2--9 and has the best antimicrobial activity at near pH 6.0; Na+, K+ had not any effect on the antibacterial activity, the effect of Mg2+, Ca2+ on the antibacterial activity was constantly weakening along with the increase of the ion concentration, the effect of Fe3+, Al3+ on the antibacterial activity promoted growth at first, and then inhibited growth at last. At the same time, the antibacterial activity of Mb-Cu was unaffected by UV treatment.In the second section, we study on the antibacterial activity by use Mb-Cu compounded with chemical preservatives sodium benzoate and potassium sorbate the best proportions of three factors compounded were optimized by use of the Response Surface Methodology (RSM), and then the optimum results were analyzed by Design-Expert 8.0.6.1. The optimize proportions of the compounds for Escherichia coli are:Mb-Cu:sodium benzoate:potassium sorbate= 0.182:0.065:0.261 (mg/mL), at this time get the maximum inhibition circle diameter reached the biggest 28.18mm by the filter paper method. Staphylococcus albus are:Mb-Cu: sodium benzoate:potassium sorbate=0.330:0.309:0.144 (mg/mL), the maximum inhibition circle diameter reached the biggest 34.99mm.The effect of Mb-Cu on the growth curve of indicator bacteria showed that the lag period of Escherichia coli was extended for 0.5h and the maximum growth rate in the log phase reduces one times and the lag period of Staphylococcus albus was extended for 6.5h and the maximum growth rate in the log phase reduces 0.5 times by the addition of Mb-Cu. The results indicated that Mb-Cu mainly inhibited the log phase of Escherichia coli and mainly inhibited the lag period of Staphylococcus albus.Mb-Cu can increase the conductivity of culture medium of Escherichia coli and Staphylococcus albus by electrical conductivity method. The results showed that the indicator bacteria cell membrane was damage and increase the permeability, which lead to cytoplasm leakage and cytoplasm within the charged material to the indicator bacteria in the culture medium, leading fluid conductivity increased.The results of indicator bacteria bacterial protein SDS-PAGE electrophoresis analysis by addition Mb-Cu showed that affected the synthesis or expression of protein, the protein decreased or disappeared.The antibacterial mechanism of Mb-Cu against Escherichia coli and Staphylococcus albus was that Mb-Cu acted on the cell membrane and destroy of cell integrity, which induced membrane permeability increased. Thus the charged substance and proteins in the cell to leak out of it, and Mb-Cu also to inhibit the synthesis or expression of intracellular proteins and finally causes the cell death.