구름버섯 균사체 배양액 추출물의 생리활성 및 약용버섯 균사체의 배양특성

Abstract

Species of the mushroom mycelium were grown in a defined synthetic liquid medium(YM) and dilute citrus extracts for culture characteristics, antioxidant activity, antibacterial activity, superoxide radical scavenging activity. Coriolus versicolor, Lentinus edodes, Cordyceps militaris, Agaricus blazei, followed by fast growth and Sparassis crispa was the late for 15 days. After culture weight of dried cells was Lentinus edodes, Ganoderma lucidium, Coriolus versicolor, and exo-polysaccharide was formed by Lentinus edodes, Hericium erinacium, Coriolus versicolor, Cordyceps militaris. The antibacterial activity of the culture extracts obtained from the YM medium was Sparassis crispa> Coriolus versicolor> Phellinus linteus> Agaricus blazei> Hericicum eninacium and the culture extracts obtained from the dilute citrus extracts medium was got similar results. The culture extracts obtained from the Sparassis crispa showed 80% of the DPPH radical scavenger activity in 1mg/mL and all culture extracts showed 40% of the DPPH radical scavenger activity over 5mg/mL. Also culture extracts obtained from the Sparassic crispa showed over 70% of the enzyme activity by superoxide radical scavenging activity. Overall culture extracts showed BHA, synthetic anti oxidizing agent, higher than the enzyme activity. Coriolus versicolor was grown in a defined YM medium and citrus extracts, and the culture extracts were examined for antioxidant activity, nitrite scavenging activity, and in vitro anticancer activity against HeLa (female cervix adenocarcinoma), PC-3 (male prostate adenocacinoma), HepG2 (human hepatoblastoma) and A-549 (male lung carcinoma) cells. Whereas the culture extracts obtained from the YM medium and the un-inoculated citrus extract showed 60 and 22% of the DPPH radical scavenger activity, the culture extracts obtained from the citrus medium exhibited antioxidant activity up to 89%. The nitrite scavenging activity of the culture extracts obtained from the citrus medium and the YM medium, and the un-inoculated citrus extract at pH 1.2 were up to 67, 55, and 34%, respectively. the culture extract obtained from the YM medium inhibited the growth of HeLa, PC-3, HepG2, and A-549 up to 66, 23, 18, 10% at 48 hr of incubation, respectively; however, the culture extract obtained from the citrus medium inhibited the growth of HeLa, PC-3, HepG2, and A-549 up to 75, 82, 55, and 82%, respectively. As a negative control, the un-inoculated dilute citrus extract was examined in the same way and inhibited the growth of HeLa, PC-3, and HepG2 cells 20, 6, and 15% at 48 hr of incubation, respectively; the inhibition of A-549 cell growth was negligible. These results clearly showed that the fermentation of Coriolus versicolor in the dilute citrus extracts rather than in the defined YM medium significantly enhanced the anticancer activity, antioxidant activity, and nitrite scavenging activity. The ethyl acetate extracts from the liquid cultures of Coriolus versicolor, Phellinus linteus, and Hericium erinaceus showed significant antibacterial activities against E. coli K88, E. coli K99, E. coli 987P, and Salmonella typhimurium 14058 which are known to cause bacterial diarrhea in Korean house pigs and chicken. Of these extracts, Coriolus versicolor extract showed the highest antibacterial activity. In addition, these extracts also showed significant growth inhibition against Staphylococcus aureus CARM3230 and E. coli CARM1381 which are known as kanamycin and ampicillin-resistant strains, with the highest activity by the Coriolus versicolor extract. These results showed that the mushrooms extracts could be developed as a livestock feed additives that can replace or, at least, reduce the intensive use of commercial antibiotics, and also could be a good source for the development of a new antibacterial agent. The liquid culture extract of Coriolus versicolor grown on citrus medium was prepared by directly boiling the whole culture broth on day 7 of the growth and then removal of mycelial debris through filtration. This extract was further extracted with equal volume of ethyl acetate (1:1, v/v). The ethyl acetate extracts showed significant antibacterial activities against Staphylococcus aureus CCARM3230 and Pseudomonas aeruginosa CCARM2171, which are resistant to several antibiotics. The most active fraction was eluted from a silica gel column with a mixture of dichloromethane and methanol (9:1, v/v) and the purity of this active substance was confirmed by HPLC analysis. The results suggest that the purified active substance could be a good source for the development of a new antimicrobial agent, especially for the treatment of antibiotic resistant bacteria. The optimal culture condition of Phellinus linteus, Cordyceps militaris, Coriolus versicolor, Sparassis crispa, Agaricus blazei, Inonotus obliquus, Lentinus edodes, Hericium erinacium, Ganoderma lucidium showed about 25℃, pH 4.5～6.5. Manitol, mannose, sucrose, soluble starch, maltose, glucose has been as a variety of carbon source and yeast extract, malt extract has been used to nitrogen source, mostly K2HPO4 was used as a mineral source. Lentinus edodes has been used to CaCl2 for mineral source. Mixing culture condition of Phellinus linteus and Coriolus versicolor showed that after 3 days of cultured respectively and the rate of mixing 1:2 have been high level of growth and production of exo-polysaccarides. The antibacterial activity of the culture extracts obtained from the YM medium and the mixing culture extracts obtained from the dilute citrus extracts medium were similar results.