尖孢镰刀菌LD105产抑菌多糖固态发酵条件优化
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摘要
以云南昆明玛卡根际土壤中分离到的尖孢镰刀菌LD105作为发酵菌种,以多糖产量和抑菌率为评价指标,用响应面法优化尖孢镰刀菌产抑菌多糖固态发酵工艺。通过单因素试验,筛选出发酵时间、初始含水量、蔗糖和KH_2PO_4添加量4个显著影响因素,研究其交互作用,优化得出最佳发酵工艺条件:发酵时间5 d、初始含水量54%、发酵温度28℃、初始pH 6、蔗糖添加量3%、KH_2PO_4添加量0.3%、接种量10%、蛋白胨添加量1.5%、装料量10 g(250 mL锥形瓶为发酵容器)。在此优化条件下,尖孢镰刀菌固态发酵产多糖抑菌率和多糖产量分别为37.6%和30.2 mg/g,抑菌率和多糖产量比优化前分别提高了1.8倍及1.5倍。
Response surface methodology was employed to optimize the solid-state fermentation process of Fusarium oxysporum LD105, isolated from maca rhizosphere soil in Kunming, Yunnan Province, for improved production of antibacterial polysaccharides. Polysaccharide yield and antibacterial activity were used as response variables. Using one-factor-at-a-time method, fermentation time, initial moisture content, sucrose and KH_2PO_4 concentration were identified as main factors that significantly influence the responses. The three-dimensional response surfaces were generated to study the interaction among the four factors. The results showed that the optimal fermentation conditions were as follows: fermentation time 5 d; sucrose concentration 3%; fermentation temperature 28 ℃, initial moisture content 54%, KH_2PO_4 concentration 0.3%, inoculum size 10%, initial medium pH 6, peptone concentration 1.5%, and 10 g of medium contained in a 250 mL conical flask. Under these conditions, the percentage inhibition of Escherichia coli and the polysaccharide yield were 37.6% and 30.2 mg/g, which were increased by 2.8 and 2.5 folds as compared to that obtained using the unoptimized medium, respectively.
Response surface methodology was employed to optimize the solid-state fermentation process of Fusarium oxysporum LD105, isolated from maca rhizosphere soil in Kunming, Yunnan Province, for improved production of antibacterial polysaccharides. Polysaccharide yield and antibacterial activity were used as response variables. Using one-factor-at-a-time method, fermentation time, initial moisture content, sucrose and KH_2PO_4 concentration were identified as main factors that significantly influence the responses. The three-dimensional response surfaces were generated to study the interaction among the four factors. The results showed that the optimal fermentation conditions were as follows: fermentation time 5 d; sucrose concentration 3%; fermentation temperature 28 ℃, initial moisture content 54%, KH_2PO_4 concentration 0.3%, inoculum size 10%, initial medium pH 6, peptone concentration 1.5%, and 10 g of medium contained in a 250 mL conical flask. Under these conditions, the percentage inhibition of Escherichia coli and the polysaccharide yield were 37.6% and 30.2 mg/g, which were increased by 2.8 and 2.5 folds as compared to that obtained using the unoptimized medium, respectively.
引文
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[2]ENGLAND M W,SATO T,URATA C,et al.Transparent gel composite films with multiple functionalities:long-lasting antifogging,underwater superoleophobicity and anti-bacterial activity[J].Journal of Colloid&Interface Science,2017,505:566.DOI:10.1016/j.jcis.2017.06.038.
[3]ANANTHI S,RAGHAVENDRAN H R,SUNIL A G,et al.In vitro antioxidant and in vivo anti-inflammatory potential of crude polysaccharide from Turbinaria ornata (Marine Brown Alga)[J].Food&Chemical Toxicology,2010,48(1):187-192.DOI:10.1016/j.fct.2009.09.036.
[4]SAMAVATI V,MANOOCHEHRIZADE A.Polysaccharide extraction from Malva sylvestris and its anti-oxidant activity[J].International Journal of Biological Macromolecules,2013,60(6):427-436.DOI:10.1016/j.ijbiomac.2013.04.050.
[5]孟翔鹏,马琳.食药用真菌的研究进展及其应用前景展望[J].中国现代中药,2009,11(10):7-10.DOI:10.3969/j.issn.1673-4890.2009.10.003.
[6]DUVNJAK D,PANTI?M,PAVLOVI?V,et al.Advances in batch culture fermented Coriolus versicolor medicinal mushroom for the production of antibacterial compounds[J].Innovative Food Science&Emerging Technologies,2016,34:1-8.DOI:10.1016/j.ifset.2015.12.028.
[7]SHARMA S,KHANNA P K,KAPOOR S.Optimised isolation of polysaccharides from Lentinula edodes strain NCBI JX915793 using response surface methodology and their antibacterial activities[J].Natural Product Research,2016,30(5):616-621.DOI:10.1080/14786419.2015.1030741.
[8]ELLIOTT M L,DES JARDIN E A,O’DONNELL K,et al.Fusarium oxysporum f.sp.palmarum,a novel forma specialis causing a lethal disease of Syagrus romanzoffiana and Washingtonia robusta in Florida[J].Plant Disease,2010,94(1):31-38.DOI:10.1094/PDIS-94-1-0031.
[9]PETKAR A,LANGSTON,STEVENSON,et al.Sensitivity of Fusarium oxysporum f.sp.niveum to prothioconazole and thiophanatemethyl and gene mutation conferring resistance to thiophanatemethyl[J].Plant Disease,2017,101(2):366-371.DOI:10.1094/PDIS-09-16-1236-RE.
[10]李绍平,冯昆,孟兰贞,等.尖孢镰刀菌提取物及其应用:2013135119 A1[P].2013[2017-12-08].
[11]ALI S S,VIDHALE N N.Protease production by Fusarium oxysporum in solid-state fermentation using rice bran[J].American Journal of Microbiological Research,2013,1(3):45-47.DOI:10.12691/ajmr-1-3-2.
[12]鲁群.二十五株丝状真菌及其次级代谢产物的分离纯化与鉴定[D].广州:华南理工大学,2015.
[13]WU S,JIN Z,TONG Q,et al.Sweet potato:a novel substrate for pullulan production by Aureobasidium pullulans[J].Carbohydrate Polymers,2009,76(4):645-649.DOI:10.1016/j.carbpol.2008.11.034.
[14]邵丽.产胞外多糖乳杆菌的筛选及其多糖的分离、结构和生物活性研究[D].无锡:江南大学,2015.
[15]WANG L Y,CHEONG K L,WU D T,et al.Fermentation optimization for the production of bioactive polysaccharides from Cordyceps sinensis fungus UM01[J].International Journal of Biological Macromolecules,2015,79(2/3):180-185.DOI:10.1016/j.ijbiomac.2015.04.040.
[16]LI Z,YE X,CHEN P,et al.Antifungal potential of Corallococcus sp.strain EGB against plant pathogenic fungi[J].Biological Control,2017,110:10-17.DOI:10.1016/j.biocontrol.2017.04.001.
[17]CASARIL,BENTOKASUYA K B P,MEGUMIVANETTI M C,et al.Antimicrobial activity and mineral composition of shiitake mushrooms cultivated on agricultural waste[J].Brazilian Archives of Biology&Technology,2011,54(5):991-1002.DOI:10.1590/S1516-89132011000500017.
[18]安超,马赛箭,薛文娇,等.无机氮源对出芽短梗霉发酵普鲁兰多糖分子量的影响[J].现代食品科技,2017,33(5):213-219.DOI:10.13982/j.mfst.1673-9078.2017.5.034.
[19]李晓秀,张盼,刘琬瑜,等.玉米秸秆降解真菌的筛选及鉴定[J].饲料工业,2017(3):37-42.
[20]YAN Y,LI W P,GAO W J,et al.Application of rDNA ITS sequence analysis in fungus identification[J].Chinese Journal of Health Laboratory Technology,2008,18(10):1958-1961.
[21]陈瑜,梁运祥,胡咏梅,等.固体发酵灵芝菌丝多糖的提取及组分分析[J].中国食用菌,2007,26(3):34-37.DOI:10.3969/j.issn.1003-8310.2007.03.012.
[22]SHI M,YANG Y,LI Y,et al.Optimum condition of ecologic feed fermentation by Pleurotus ostreatus using soybean curd residue as raw materials[J].International Journal of Biology,2011,3(4):2-12.DOI:10.5539/ijb.v3n4p2.
[23]TENGERDY,ROBERT P.Solid substrate fermentation[J].Trends in Biotechnology,1985,3(4):96-99.DOI:10.1016/0167-7799(85)90092-7.
[24]HSIEH C,YANG F C.Reusing soy residue for the solid-state fermentation of Ganoderma lucidum[J].Bioresource Technology,2004,91(1):105-109.DOI:10.1016/S0960-8524(03)00157-3.
[25]孙秋芳.树舌灵芝发酵条件的优化及发酵产物抑菌活性的研究[D].桂林:广西师范大学,2011.
[26]朱强,夏艳秋,陈爱芹,等.响应面法优化灵芪菌质多糖固态发酵条件[J].中国生物工程杂志,2011,31(12):99-103.DOI:10.13523/j.cb.20111216.
[27]SON S,KIM H,CHOI G,et al.Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against phytophthora infestans[J].Journal of Applied Microbiology,2008,104(3):692-698.DOI:10.1111/j.1365-2672.2007.03581.x.
[28]蒲立柠,陈光静,阚建全.响应面试验优化青稞麸皮薏仁红曲霉发酵工艺[J].食品科学,2017,38(2):264-270.DOI:10.7506/spkx1002-6630-201702041.
[29]李作美,吴珊珊,陈佳.大秃马勃菌丝体多糖的发酵条件优化及其抑菌作用[J].中国酿造,2016,35(8):120-123.DOI:10.11882/j.issn.0254-5071.2016.08.027.
[30]LIU Z,YAO L,FAN C.Optimization of fermentation conditions of pectin production from Aspergillus terreus and its partial characterization[J].Carbohydrate Polymers,2015,134(17):627-634.DOI:10.1016/j.carbpol.2015.08.032.