一株桃树内生真菌Pestalotiopsis vismiae N1培养条件优化与抗氧化、抗菌活性分析
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摘要
对一株内生真菌韦司梅拟盘多毛孢(Pestalotiopsis vismiae)的培养条件进行探究,在此基础上进一步对其发酵提取物抗氧化、抗菌活性进行分析.通过测定菌丝的生长速度寻找菌丝培养适宜的温度、碳源、氮源和pH,并采用正交试验优化培养条件;通过测定菌株深层发酵液提取物对羟基和DPPH自由基的清除率以分析其抗氧化活性;通过测定菌株深层发酵液提取物对多种病原细菌抑菌圈大小的影响来分析其抗菌活性.结果表明,韦司梅拟盘多毛孢菌丝最适宜培养温度为25℃,在该温度培养条件下菌丝生长最佳碳源为蔗糖,最佳氮源为酵母提取粉,最佳初始pH为6.5,这3种因素的显著性差异大小顺序为碳源>氮源> pH;菌株深层发酵液提取物具有良好的抗氧化活性,其对羟基自由基和DPPH自由基清除率的EC50分别为1.19 mg/mL和0.56 mg/mL;菌株发酵液提取物对铜绿假单胞杆菌、金黄色葡萄球菌均具有一定的抑制作用;深层发酵液提取物抗氧化和抗菌活性具有一定的稳定性.本研究结果可为深入开发利用该真菌资源提供科学依据.(图12表3参26)
In this study, the culture conditions of endophytic fungus Pestalotiopsis vismiae were investigated, and the antioxidant and antibacterial activities were analyzed to provide a basis for further development and utilization of P. vismiae.The mycelial growth rate was determined to explore the suitable temperature, carbon source, nitrogen source, and pH for mycelial growth, and the orthogonal test was performed to optimize the culture conditions. The antioxidant activity of the extract from the deep fermentation broth was analyzed by measuring the hydroxyl radical and DPPH radical scavenging rates.The antibacterial activity of the extract from the deep fermentation broth was studied by measuring the size of the inhibition zone for various pathogenic bacteria. The results showed that the optimum growth temperature for P. vismiae was 25 ℃, the best carbon source for mycelial growth at 25 ℃ was sucrose, the optimum nitrogen source was yeast extract, and the optimum initial pH was 6.5. The order of the significant differences among the three factors was carbon source > nitrogen source > pH.The submerged fermentation broth extract of P. vismiae had good antioxidant activity, and the EC50 of the hydroxyl radical and DPPH radical scavenging rates was 1.19 mg/mL and 0.56 mg/mL, respectively. The strain fermentation broth extract had certain inhibitory effects on Pseudomonas aeruginosa and Staphylococcus aureus. The antioxidant and antibacterial activity of P. vismiae had a certain stability. The results of this study provided a scientific basis for the development and utilization of these fungal resources.
In this study, the culture conditions of endophytic fungus Pestalotiopsis vismiae were investigated, and the antioxidant and antibacterial activities were analyzed to provide a basis for further development and utilization of P. vismiae.The mycelial growth rate was determined to explore the suitable temperature, carbon source, nitrogen source, and pH for mycelial growth, and the orthogonal test was performed to optimize the culture conditions. The antioxidant activity of the extract from the deep fermentation broth was analyzed by measuring the hydroxyl radical and DPPH radical scavenging rates.The antibacterial activity of the extract from the deep fermentation broth was studied by measuring the size of the inhibition zone for various pathogenic bacteria. The results showed that the optimum growth temperature for P. vismiae was 25 ℃, the best carbon source for mycelial growth at 25 ℃ was sucrose, the optimum nitrogen source was yeast extract, and the optimum initial pH was 6.5. The order of the significant differences among the three factors was carbon source > nitrogen source > pH.The submerged fermentation broth extract of P. vismiae had good antioxidant activity, and the EC50 of the hydroxyl radical and DPPH radical scavenging rates was 1.19 mg/mL and 0.56 mg/mL, respectively. The strain fermentation broth extract had certain inhibitory effects on Pseudomonas aeruginosa and Staphylococcus aureus. The antioxidant and antibacterial activity of P. vismiae had a certain stability. The results of this study provided a scientific basis for the development and utilization of these fungal resources.
引文
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13 Zhang Y,Liu X,Wang Y,Jiang P,Quek SY.Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli,and Staphylococcus aureus[J].Food Control,2016,59:282-289
14 Hu D,Wang H,Wang L.Physical properties and antibacterial activity of quaternized chitosan/carboxymethyl cellulose blend films[J].LWT-Food Sci Technol,2016,65:398-405
15郑艳,戴婧婧,管玉鑫,姜莹莹,邢晴晴.凤丹内生菌的分离鉴定及抑菌活性研究[J].中国中药杂志,2016,41(1):45-50[Zheng Y,Dai JJ,Guan YX,Jiang YY,Xing XX.Isolation,identification and inhibition activity of endophytes in Fengdan[J].Chin J Trad Chin Med,2016,41(1):45-50]
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20 Manoharan S,Guillemin GJ,Abiramasundari RS,Essa MM,Akbar M,Akbar MD.The role of reactive oxygen species in the pathogenesis of alzheimer’s disease,parkinson’s disease,and huntington’s disease:a mini review[J].Oxid Med Cell Long,2016,2016(4):8590578
21韩青松,简永利,涂宜强,高永安,宋显章,涂国众,段龙川.绿脓杆菌研究进展[J].畜牧与饲料科学,2012,33(1):122-124[Han QS,Jian YL,Tu YQ,Gao YA,Song XZ,Tu GZ,Duan LC.Research advances on Pseudomonas aeruginosa[J].Animal Husbandry Feed Sci,2012,33(1):122-124]
22 Stover CK,Pham XQ,Erwin AL,Mizoguchi SD,Warrener P,Hickey MJ,Brinkman FS,Hufnagle WO,Kowalik DJ,Lagrou M,Garber RL,Goltry L,Tolentino E,Westbrock-Wadman S,Yuan Y,Brody LL,Coulter SN,Folger KR,Kas A,Larbig K,Lim R,Smith K,Spencer D,Wong GK,Wu Z,Paulsen IT,Reizer J,Saier MH,Hancock RE,Lory S,Olson MV.Complete genome sequence of Pseudomonas aeruginosa PAO1,an opportunistic pathogen[J].Nature,2000,406(6799):959-64
23徐海花,牛钟相,秦爱建.细菌耐药性研究进展[J].山东农业大学学报(自然科学版),2010,41(1):156-160[Xu HH,Niu ZX,Qin AJ.New research progress of bacterial drug-resistance[J].J Shandong AgricUniv(Nat Sci Ed),2010,41(1):156-160]
24侯若琳,刘鑫,陈良韬,李佳欢,郑明锋,傅俊生.一株韦司梅拟盘多毛孢的鉴定及其发酵液抗乳腺癌活性[J].西北农林科技大学学报(自然科学版),2019,47(3):111-120[Hou RY,Liu X,Chen LT,Li JH,Zheng MF,Fu JS.Identification of a Pestalotiopsis vismiae and anti-breast cancer activity of its fermentation broth[J].J NW A&F Univ(Nat Sci Ed),2019,47(3):111-120
25王翔林,陈晨,王冠楠.HPLC法测定胍基乙酸含量[J].黑龙江畜牧兽医,2016,2:254-257[Wang XL,Chen C,Wang GN.Determination of Glycocyamine by HPLC[J].Heilongjiang Anim Husbandry,2016,2:254-257]
26 Aree T,Jongrungruangchok S.Enhancement of antioxidant activity of green tea epicatechins inβ-cyclodextrin cavity:single-crystal X-ray analysis,DFT calculation and DPPH assay[J].Carb Polym,2016,151(20):1139-1151
2 Strobel G,Daisy B,Castillo U.Natural products from endophytic microorganisms[J].J Nat Prod,2004,67(2):257-268
3 Strobel G,Yang X,Sears J,Kramer R,Sidhu RS,Hess WM.Taxol from Pestalotiopsis microspora,an endophytic fungus of taxus wallachiana[J].Microbiology,1996,142(2):435-440
4钱一鑫,康冀川,雷帮星.一株牡蒿内生尤韦可拟盘多毛孢及其应用:中国,CN 104212723 A[P].2014-12-17[Qian YJ,Kang YC,Lei BX.An endophytes Pestalotiopsis uvicola from Artemisia Artemisia and its application:China,CN 104212723 A[P].2014-12-17]
5张旭,龙庆德,李启瑞,何明辉,唐翠娥.一株拟盘多毛孢属真菌及其用于艳山姜叶病害的鉴定方法:中国,CN106434371A[P].2017-02-22[Zhang X,Long QD,Li QR,He MH,Tang CE.A Pestalotiopsis fungus and its application in identification of leaf diseases of Alpinia zerumbet:China,CN106434371 A[P].2017-02-22]
6王雅玲,叶日英,孙力军.一种韦司梅拟盘多毛孢CAMT66351及其应用:中国,CN107034145A[P].2017-08-11[Wang YL,Ye RY,Sun LJ.A Pestalotiopsis vismiae CAMT66351 and its application:China,CN107034145A[P].2017-08-11]
7曹春蕾,崔宝凯,包海鹰.桑木层孔菌固体培养条件的研究[J].菌物学报,2011,30(3):486-490[Cao CL,Cui BK,Bao HY.Solid culture conditions of Phellinus mori[J].Mycosystema,2011,30(3):486-490]
8王建瑞,图力古尔,刘宇,鲁铁.簇生沿丝伞的生物学特性[J].菌物研究,2014,12(1):36-40[Wang JR,Tolgor B,Liu Y,Ru L.Biological characteristics of wild medicinal mushroom Naematoloma fasciculare[J].J Fungal Res,2014,12(1):36-40]
9鲁铁,图力古尔.白蜡多年卧孔菌生物学特性及驯化栽培[J].生态学报,2013,33(17):5194-5200[Lu T,Bau T.Biological characteristics and cultivation of fruit body of wild medicinal mushroom Perenniporia fraxinea[J].Acta Ecol Sin,2013,33(17):5194-5200]
10 Yin C,Fan X,Fan Z,Shi D,Gao H.Optimization of enzymesm icrowave-ult rasou nd assisted ext ract ion of Lent inus edodes,polysaccharides and determination of its antioxidant activity[J].Int JBiol Macromol,2018(111):446-454
11 Ai S,Tnabe S,Nishimura T.Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J].J Agric Food Chem,2003,51(12):3661
12孟庆龙,潘景芝,陈丽,王琦.桑黄发酵产物的抑菌作用[J].食品科学,2011,32(3):56-59[Meng QL,Pan JZ,Chen L,Wang Q.Antibacterial activity evaluation of fermentation supernatant and mycelia of Phellinus igniarius[J].Food Sci,2011,32(3):56-59]
13 Zhang Y,Liu X,Wang Y,Jiang P,Quek SY.Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli,and Staphylococcus aureus[J].Food Control,2016,59:282-289
14 Hu D,Wang H,Wang L.Physical properties and antibacterial activity of quaternized chitosan/carboxymethyl cellulose blend films[J].LWT-Food Sci Technol,2016,65:398-405
15郑艳,戴婧婧,管玉鑫,姜莹莹,邢晴晴.凤丹内生菌的分离鉴定及抑菌活性研究[J].中国中药杂志,2016,41(1):45-50[Zheng Y,Dai JJ,Guan YX,Jiang YY,Xing XX.Isolation,identification and inhibition activity of endophytes in Fengdan[J].Chin J Trad Chin Med,2016,41(1):45-50]
16陈燕飞.pH对微生物的影响[J].太原师范学院学报(自然科学版),2009,8(3):121-124+131[Chen YF.pH to uygur biology Influence[J].JTaiyuan Norm Univ(Nat Sci Ed),2009,8(3):121-124+131]
17黄年来.中国食用菌百科[M].北京:农业出版社,1997:1-448[Huang NL.Chinese Edible Fungus Encyclopedia[M].Beijng:Agricultural Publishing House,1997:1-448]
18 Miller AA,Silva TMD,Drummond GR,Sobey CG,Chrissobolis S.Reactive Oxygen Species and Cerebrovascular Diseases[M].Berlin:Springer Berlin Heidelberg,2014:1895-1924
19 Zhou D,Shao L,Spitz DR.Reactive oxygen species in normal and tumor stem cells[J].Adv Can Res,2014,122:1-67
20 Manoharan S,Guillemin GJ,Abiramasundari RS,Essa MM,Akbar M,Akbar MD.The role of reactive oxygen species in the pathogenesis of alzheimer’s disease,parkinson’s disease,and huntington’s disease:a mini review[J].Oxid Med Cell Long,2016,2016(4):8590578
21韩青松,简永利,涂宜强,高永安,宋显章,涂国众,段龙川.绿脓杆菌研究进展[J].畜牧与饲料科学,2012,33(1):122-124[Han QS,Jian YL,Tu YQ,Gao YA,Song XZ,Tu GZ,Duan LC.Research advances on Pseudomonas aeruginosa[J].Animal Husbandry Feed Sci,2012,33(1):122-124]
22 Stover CK,Pham XQ,Erwin AL,Mizoguchi SD,Warrener P,Hickey MJ,Brinkman FS,Hufnagle WO,Kowalik DJ,Lagrou M,Garber RL,Goltry L,Tolentino E,Westbrock-Wadman S,Yuan Y,Brody LL,Coulter SN,Folger KR,Kas A,Larbig K,Lim R,Smith K,Spencer D,Wong GK,Wu Z,Paulsen IT,Reizer J,Saier MH,Hancock RE,Lory S,Olson MV.Complete genome sequence of Pseudomonas aeruginosa PAO1,an opportunistic pathogen[J].Nature,2000,406(6799):959-64
23徐海花,牛钟相,秦爱建.细菌耐药性研究进展[J].山东农业大学学报(自然科学版),2010,41(1):156-160[Xu HH,Niu ZX,Qin AJ.New research progress of bacterial drug-resistance[J].J Shandong AgricUniv(Nat Sci Ed),2010,41(1):156-160]
24侯若琳,刘鑫,陈良韬,李佳欢,郑明锋,傅俊生.一株韦司梅拟盘多毛孢的鉴定及其发酵液抗乳腺癌活性[J].西北农林科技大学学报(自然科学版),2019,47(3):111-120[Hou RY,Liu X,Chen LT,Li JH,Zheng MF,Fu JS.Identification of a Pestalotiopsis vismiae and anti-breast cancer activity of its fermentation broth[J].J NW A&F Univ(Nat Sci Ed),2019,47(3):111-120
25王翔林,陈晨,王冠楠.HPLC法测定胍基乙酸含量[J].黑龙江畜牧兽医,2016,2:254-257[Wang XL,Chen C,Wang GN.Determination of Glycocyamine by HPLC[J].Heilongjiang Anim Husbandry,2016,2:254-257]
26 Aree T,Jongrungruangchok S.Enhancement of antioxidant activity of green tea epicatechins inβ-cyclodextrin cavity:single-crystal X-ray analysis,DFT calculation and DPPH assay[J].Carb Polym,2016,151(20):1139-1151