| Auricularia auricula is well known for black, but there is sparse for the characterization and biological activity studies on A. auricula melanin. Melains are not only a kind of photoprotectants, chelators, bio-antioxidants and immuno-stimulators but also a kind of amorphous organic semiconductors, photoconductors and electrical conductors. They have many bio-activities such as anti-venom, antitumor, inhibiting HIV replication and protection against Parkinson disease. So they have a vast range of prospects for making use in functional food industry, cosmetic, medicine, pharmacy, bioelectronics and other fields. At present, the production of melanin is mainly rely on the synthesis, and natural melanin that is got from plants and animals can not meet the actual demand because of seasonal, climatic and geographical constraints. A. auricula is a precious edible fungus both for medicine and food. A. auricula has widely application and high security. To study the extraction, purification and biological activity of A. auricula melanin is of great importance in theoretical and practical. It can improve its added-value and deep processing, and it is also useful for further investigations of melanin. The main results were as follows:1. A. auricula melanin was extracted with NaOH and HCl. Purified melanin was obtained by using strong acid hydrolysis and washing by organic solvents. It was characterized by chemical tests, ultraviolet-visible (UV) absorption spectrum, infrared (IR) spectroscopy, electron paramagnetic resonance (EPR) and elemental analysis. The melanin was insoluble in both water and organic solvents, and dissolved in1.5mol/L NaOH. The overall characteristic absorption peak was showed at210nm, and the log of optical density of the melanin solution when plotted against wavelength produces a linear curve with negative slopes of-0.0028. The IR spectrum of the melanin exhibited broad absorption band at3287.6cm-1and1619.4cm-1. The EPR spectrum was a typical single first derivative spectrum, and the melanin did not have hyperfine structure. The peak at2.0042(G value) of the pigment indicated it is melanin. The S:N of the melanin is0.08.2. The biological methods of reporter agar diffusion assay and extraction of violacein were used to determine the capacity of QSI for A. auricula melanin. The results showed that A. auricula melanin did not inhibit the growth of C. violaceum CV026, but it can inhibit the production of violacein. Inhibition with the concentration of the melanin showed significant dose-effect relationship.3. The swarming motility in P. aeruginosa PAO1was recognized as QS regulation of behavior. It was screened using agar diffusion method. The results showed that A. auricula melanin reduced swarming motility in P. aerugionsa PAO1at sub-MIC. As the concentration increases, the inhibition rate was from21.06%to94.25%.4. The influence on biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin was checked. The results of qualitative and quantitative methods showed that the A. auricula melanin can inhibit biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3at sub-MIC. Furthermore, we studied the inhibition of biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin at different concentrations and found that inhibition with the concentration of the melanin showed significant dose-effect relationship. The percentage inhibition of biofilm formation by A. auricula melanin at a concentration of80μg/mL were73.78%,53.01%and55.58%, respectively.5. The influence on mature biofilm of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3by A. auricula melanin with potassium sorbate, ε-polylysine and Nisin was checked. Sub-MIC of A. auricula melanin can reduce the minimum biofilm bacterium elimination concentration (MBEC) of the preservative to E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3significantly. The confocal laser scanning microscopy (CLSM) images showed that the80μg/mL of A. auricula melanin can damage the mature biofilm of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3. The failure rate were41.99%,36.28%and40.25%, respectively.6. Using DPPH, superoxide and hydroxyl radical scavenging assay, the A. auricula melanin exhibited a moderate antioxidant activity. It is clearly demonstrated that free radical scavenging increases with increasing melanin concentration. A. auricula melanin had strong scavenging activity on hydroxyl radical.Therefore, A. auricula melanin that was extracted with NaOH and HC1and purified by using strong acid hydrolysis and organic solvent was eumelanins. It had the typical characteristics of the natural melanin (including solubility, UV spectroscopy, IR spectroscopy, EPR). Purified A. auricula melanin can inhibit the production of violacein of C, violaceum CV026, reduce swarming motility in P. aerugionsa PAO1and inhibit biofilm formation of E. coli K-12, P. aeruginosa PAO1and P. fluorescens P-3. A. auricula melanin also can scavenge DPPH, superoxide and hydroxyl. A. auricula melanin can be used as a natural QSI and antioxidant in food and pharmaceutical industries. |
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