蛹虫草SOD高活性菌株筛选及液体培养条件优化
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摘要
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p H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 m L/250 m L和p H 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/m L;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 m L working volume in a 250 m L flask,and at p H = 5. 5. Under this condition,the SOD activity was 19. 74 U/m L( 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.
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 m L working volume in a 250 m L flask,and at p H = 5. 5. Under this condition,the SOD activity was 19. 74 U/m L( 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.
引文
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[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.
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[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
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[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.
[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 liquidcultured 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.
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