Production Of Phenoxazinone Pigments By Pycnoporus Sanguineus And Their Antibacterial Activity

As a kind of natural pigments, the phenoxazinones have some pharmacological properties and biological activities, like antibacterial, antiviral, anticancer etc, and used as antibiotics for research and clinical treatment. It mainly derived from microbial metabolic synthesis, for instance, Streptomycete species. The genus Pycnoporus, a representative white-rot filamentous fungus which can synthetise various phenoxazinones, there are some significance and potential applications for the study on the phenoxazinones pigment fermentation and their antibacterial activity.In this study, a strain of high pigment production fungi was isolated. Investigated pigment production with agricultural wastes as the substrate of solid-state fermentation(SSF), pigment extract, the fermentation condition, as well as the antibacterial activity of pigment and component analysis. The results were as follows:(1) A strain of high pigment production fungi was isolated through plate screening and fermentation screening, and was identified as Pycnoporus sanguineus SYBC-L7 based on morphological characteristics and ITS-5.8S r RNA gene sequence analysis.(2) Using methanol as the extraction solvent, the pigment can be maximally extracted from the solid culture at 35 °C after 40 min with water bath and the material to liquid ratio was 0.3:15(g: m L). When extracted with twice at the same condition, the ration of extraction could reach 95%.(3) With HPLC method, the pigment sample contained the phenoxazinone constituents of Cinnabarinic acid at 36.502 min, while its concentration was low, and this result was proved by LC-MS-MS(m/z 301.0). At the same time, we inferred out the possible constituents of Cinnabarin(m/z 287.0), Tramesanguin(m/z 285.0) and 2-Amino-9-formylphenoxazone-1-carbonic acid(m/z 285.0) through MS results(4) The culture conditions were optimized by “one factor at a time” method and orthogonal experiment. With the optimized culture media of 3.0 g wood chips mixed with 5.6 m L nutrition solution(g·L-1): glucose 30, ammonium tartrate 20, Mg SO4·7H2O 2, KH2PO4 1, Na2HPO4 0.2, Mn SO4 0.034, and initial p H was 6. When inoculated 2 days’ seed with 5 m L, and cultured at 30 °C, 75%- 80% relative humidity, without light for 10 days, the pigment yield was 4.323 mg·gdfs-1, which was 1.5 times of previous conditions(1.578 mg·gdfs-1).(5) Pigment produced by P. sanguineus SYBC-L7 with solid-state fermentation showed antibacterial activity on some Gram-positive bacillus bacteria, like B. cereus SYBC-hb8, B. pumilus SYBC-hb4, B. thuringiensis SYBC-hb7, etc, and emerged inhibition zone at the concentration of 0.0216 mg·m L-1, while only at the concentration of 0.0432 mg·m L-1, the inhibition zone was appeared for some Gram-positive bacillus and cocci bacteria, like B. subtilis SYBC-hb5, Lysinibacillus sp H021-S8, Staphylococcus kloosii H008-B4, S. aureus and so on. Furthermore, the pigment showed significant inhibitory effect on B. pumilus SYBC-hb4, B. subtilis SYBC-hb5, B. licheniformis SYBC-hb2 and B. amyloliquefaciens Y1-A3 in liquid culture, and the inhibitory ratio was near 98% when the pigment concentration was 4.32 μg·m L-1 in the culture medium. However, the pigment was no antibacterial activity for the Gram-negative bacillus bacteria, like Escherichia coli J159 and Strratia sp. L1, etc.