建立了一种较为系统的蛹虫草液体发酵产SOD的培养条件。以4种来源蛹虫草(Cordyceps militaris)菌株YCC-B、YCC-C、YCC-W、YCC-Y为研究对象,以生物量和SOD活性为指标,筛选出SOD高活性菌株。之后优化SOD高活性菌株的液体发酵条件,以生物量、SOD清除超氧阴离子能力(清除率)、酶活力和比活力为测定指标,选取接种量、装液量和pH展开3因素3水平的正交实验。经筛选,得到SOD高活性菌株为YCC-W。通过优化YCC-W的液体发酵条件,得出最佳组合为接种量4%,装液量100 mL/250 mL和pH 5.5。SOD清除率可提高41.17%,达到54.84%;SOD酶活力可提高42.04%,达到19.74 U/mL;比活力可提高44.23%,达到56.34 U/mg。该研究提供了通过蛹虫草菌丝体发酵产SOD的方法,在替代传统动物血液获取SOD上具有一定的应用前景;通过系统优化发酵条件SOD活性得到显著提升,通过继续扩大实验规模,有望用于SOD的规模化生产。
To establish a fermentation process to produce SOD by Cordyceps militaris, the biomass and SOD activity of the mycelia from four strains of C. militaris,YCC-B, YCC-C, YCC-W, and YCC-Y, were examined. Of which, C. militaris YCC-W produced the highest activity of SOD. The optimized fermentation condition for producing SOD from YCC-W was as follows: 4% inoculum, 100 mL working volume in a 250 mL flask, and at pH=5.5. Under this condition, the SOD activity was 19.74 U/mL (increased 42.04%) and the specific activity was 56.34 U/mg (enhanced by 44.23%). Besides, the removal rate of SOD increased by 41.17% and reached 54.84%. In conclusion, this study provides a new method for producing SOD by C. militaris fermentation, which shows a certain application prospect in replacing traditional animal blood to obtain SOD.
[1] 李玉,李泰辉,杨祝良,等.中国大型菌物资源图鉴[M].郑州:中原农民出版社,2015,1 349.
[2] 万琴,刘向阳,王松涛等.蛹虫草液体发酵及其应用的研究进展[J].食品与发酵科技,2017,53(1):88-91;114.
[3] LIN Liangtzung, LAI Yingjang, WU Sheching, et al.Optimal conditions for cordycepin production in surface liquid-cultured Cordyceps militaris treated with porcine liver extracts for suppression of oral cancer[J].Journal of Food and Drug Analysis,2018,26(1): 135-144.
[4] LI Deng, ZHOU Tongyong, PI Li, et al. Optimization of microwave-assisted extraction of cordycepic acid and cordycepin from cultured Cordyceps militaris by response surface methodology [J].Asian Journal of Chemistry,2013,25(14):8 065-8 071.
[5] ZHAO Huajie, LAI Qiangqiang, ZHANG Jianjun, et al. Antioxidant and hypoglycemic effects of acidic-extractable polysaccharides from Cordyceps militaris on type 2 diabetes mice[J].Oxidative Medicine and Cellular Longevity,2018.
[6] KWON H K,SONG M J,LEE H J,et al. Pediococcus pentosaceus-fermented Cordyceps militaris inhibits inflammatory reactions and alleviates contact dermatitis[J]. BMC Complementary and Alternative Medicine,2018,18 (1):75.
[7] XIA Yongliang,LUO Feifei,SHANG Yanfang,et al.Fungal cordycepin biosynthesis is coupled with the production of the safeguard molecule pentostatin[J].Cell Chemical Biology,2017,24(12):1 479-1 489.
[8] CHEN Lin, LIU Yuetao, GUO Qingfeng, et al. Metabolomic comparison between wild Ophiocordyceps sinensis and artificial cultured Cordyceps militaris[J].Biomedical Chromatography,2018,32(2):4 279.
[9] 都兴范,李军,米锐,等.蛹虫草和冬虫夏草主要活性成分含量比较[J].食用菌,2010,32(6):61-62.
[10] 文庭池,查岭生,康冀川,等.蛹虫草研究和开发过程中的一些问题和展望[J].菌物学报,2017,36(1):14-27.
[11] WINKLER D.Yartsa Gunbu (Cordyceps sinensis) and the fungal commodification of Tibet’s rural economy[J].Economic Botany, 2008,62(3):291-305.
[12] SUNG G H,HYWEL-JONES N L,SUNG J M,et al.Phylogenetic classification of Cordyceps and the clavicipitaceous fungi[J].Studies in Mycology,2007,57:5-59.
[13] 贺佳.固体培养蛹虫草中SOD的提取分离和性质研究[D].西安:西北大学,2014.
[14] KANG D H,KANG S W.Targeting cellular antioxidant enzymes for treating atherosclerotic vascular disease[J].Biomolecules and Therapeutics,2013,21(2):89-96.
[15] OZTURK P,ARICAN O,BELGE K E,et al.Oxidative stress in patients with scalp seborrheic dermatitis[J].Acta Dermatovenerologica Croatica,2013,21(2): 80-85.
[16] 王依屹,张珏,鲁传翠,等.超氧化物歧化酶及游离脂肪酸检测在胰岛素抵抗疾病中的应用[J].标记免疫分析与临床,2017,24 (3):247-249;289.
[17] PASTORINI S,MARINO G,BRIGATO R,et al.The therapy of plastic penile induration using superoxide dismutase per os and injection combined with vasoactive intracavernous pharmacotherapy[J].Minerva Urologica e Nefrologica,1991,43(2):75-78.
[18] BERNIER M,MANNING A S,HEARSE D J.Reperfusion arrhythmias: Dose-related protection by anti-free radical interventions[J]. American Journal of Physiology,1989,256(5):1 344-1 352.
[19] 贺阳,文连奎.越橘叶超氧化物歧化酶在食品中的应用[J].食品研究与开发,2012,33 (11):72-74.
[20] 李万芳,隆毅,刘亚鲁,等.人工栽培北虫草中超氧化物歧化酶提取工艺的优化实验[J].抗感染药学,2017,14(4):730-732.
[21] 王奇,薜阳.野生蛹虫草与培植蛹虫草SOD酶活力的对比研究[J].安徽农业科学,2008,36 (20):8 649;8 722.
[22] 梁迪思,王飞,郑家概,等.北虫草不同部位超氧化物歧化酶(SOD)酶活力的比较[J].辽宁中医药大学学报,2012,14(2):183-184.
[23] DONG Caihong,GUO Suping,WANG Wenfeng,et al.Cordyceps industry in China[J].Mycology,2015,6(2):121-129.
[24] WHITE T J, BRUNS T, LEE S, et al. Analysis of phylogenetic relationships by amplification and direct sequencing of ribosomal RNA genes[J]. PCR Protocols: A Guide to Methods and Applications, 1990, 315-322.
[25] 文庭池.蛹虫草高产虫草菌素的深层培养工艺研究[D].贵阳:贵州大学,2006.
[26] 杜琳,邱芳萍,邢燕,等.蛹虫草菌丝体中SOD提纯工艺及鉴定[J].食用菌学报,2013,20 (4):49-54
[27] 韩少华,朱靖博,王妍妍.邻苯三酚自氧化法测定抗氧化活性的方法研究[J].中国酿造,2009(6):155-157.
[28] 强伟,王洪伦,周昌范,等.考马斯亮蓝G-250染色法测定柠条锦鸡儿种子中可溶性蛋白含量[J].氨基酸和生物资源,2011,33(3):74-76.
[29] 张姝,张永杰,BHUSHAN S,等.冬虫夏草菌和蛹虫草菌的研究现状、问题及展望[J].菌物学报,2013,32 (4):577-597.
[30] XIONG Chenghui,XIA Yongliang,ZHENG Peng,et al.Developmental stage-specific gene expression profiling for a medicinal fungus Cordyceps militaris [J].Mycology,2010,1(1):25-66.
[31] 刘晓红,宋洁,栾宏伟,等.鲜蛹虫草与干蛹虫草抗氧化活性和活性物质差异研究[J].中国食用菌,2018,37(6):56-63.
