【作者】 刘姣英；

【导师】 吕作舟；

【作者基本信息】 华中农业大学 ， 微生物学， 2007， 硕士

【摘要】 长根菇（Collybia radicata）是一种珍稀药食兼用菌，味道鲜美，富含多糖、脂肪酸、蛋白质、氨基酸、矿物元素等多种营养成分，其发酵产物丰富，具有良好的抑菌功效，其多糖具有抗氧化活性、增强机体免疫活性和抑制肿瘤等作用。目前对长根菇的研究主要集中在驯化、栽培方面，而对长根菇液体发酵、发酵液抑菌活性、长根菇多糖的分离纯化及抗氧化活性方面的研究尚未见报道。本论文的主要研究内容包括：长根菇的液体培养工艺研究、发酵液抑菌活性的初步研究、多糖分离纯化及多糖的抗氧化作用研究。1．配方筛选的结果表明：在摇瓶水平上对培养基进行了单因素试验，筛选出最佳的碳源、氮源、无机盐、生长因子，采用正交试验，得出适合长根菇液体发酵培养最适培养基配方为：碳源蔗糖30g／L，氮源豆饼粉9g／L，无机盐KH₂PO₄和MgSO₄·7H₂O组合浓度为5g／L+2.5g／L，生长因子维生素B₁ 0.2g／L；单因素试验获得了最佳摇瓶培养条件是：培养温度为26℃，装液量为250 mL摇瓶装液量100mL，摇瓶转速为180r／min，接种量为10％（v／v），起始pH值5.5。验证试验表明，应用上述条件进行液体发酵，菌丝体干重达到1.514g／100mL，胞外多糖最高可达0.291g／100mL。2．长根菇发酵液对几种常见的病原菌均有一定的抑制效应。对7种供试细菌的抑菌试验结果表明，发酵液对胡萝卜软腐欧文氏菌的抑制作用最强，抑菌圈直径达4.3mm。在6种供试病原真菌菌株中抑菌试验结果表明，对稻瘟病菌的抑制作用最强，达到99％以上。稳定性试验表明，发酵时间11～13d之间抑菌活性最强；抑菌活性随温度的升高而降低，但100℃处理发酵液后抑菌活性仍高达52.804％；在pH值5.5～7.0条件下抑菌活性较高；发酵液的抑菌活性对紫外线敏感。3．抗氧化试验结果表明，长根菇不同来源的多糖（干菌丝多糖、鲜菌丝多糖、胞外多糖、菌盖多糖、菌柄多糖）和发酵原液的均有一定的抗氧化活性。各种不同来源的多糖对DPPH·均有较强的清除力，其中以干菌丝多糖清除活性最强，EC₅₀（DPPH·）值为0.394mg／mL，且随浓度的增加清除力增加。与阳性对照维生素C和维生素E相比，长根菇不同来源多糖，DPPH·清除率优于维生素E，但比维生素C差。还原力测定发现随着多糖浓度的增加，还原力增加。其中胞外多糖和干菌丝多糖还原力最大，EC₅₀（还原力）值分别为0.478mg／mL和1.068mg／mL。与阳性对照维生素C相比，还原力没有维生素C强。各种不同来源的多糖对铁离子均有较强的螯合力，且随浓度的增加铁离子螯合力增加，其中鲜菌丝多糖的铁离子螯合力最大，EC₅₀（铁离子螯合力）值为0.126mg／mL。与阳性对照EDTA相比，铁离子螯合力没有EDTA大。更多还原

【Abstract】 Collybia radicata is a rare edible and medicinal mushroom, tasty flavor, and rich inpolysaccharides, fatty acids, protein, amino acids, minerals and other nutritionalcomponents. Fermentation metabolites of Collybia radicata which has good antimicrobialactivities is very rich, and the polysaccharides of Collybia radicata have antioxidantactivity, enhance immune activity and have inhibitory effects of tumor growth. Recently,the study of Collybia radicata mainly focus on cultivation and domestication, but thestudy of liquid fermentation, antimicrobial activities of the fermentation broth, isolationand purification of Collybia radicata polysaccharides and antioxidative activities of thepolysaccharides have not been reported.The main content of this thesis includes: study on liquid culture technology ofCollybia radicata, preliminary study on antimicrobial activities of the fermentation broth,study on isolation and purification of polysaccharides and its antioxidative activitiesresearch.1. Formula screening results showed: in shaking flask, the best carbon source,nitrogen source, inorganic salts and growth factor were screened by single-factorexperiment in fermentation media. Then the optimum levels were selected by orthogonalexperiment. The fermentation media obtained to fit for Collybia radicata was: sucrose30g/L, soybean meal 9g/L, inorganic salts composition KH₂PO₄ 5g/L and MgSO₄·7H₂O2.5g/L, growth factor vitamin B₁ 0.2g/L.The optimum conditions obtained by single-factor experiment were: cultivationtemperature 26℃, loading volume 100mL of 250mL flask, rotation speed 180r/min,inoculation volume 10％（v/v） and initial pH5.5.Verification tests indicated that the above conditions were the best condition forCollybia radicata liquid fermentation, mycelium dry weight reached 1.514g/100mL andextracelluar polysaccharide were up to 0.291 g/100mL.2. The experiments of antimicrobial activities of Collybia radicata fermentationbroth showed that it had some inhibitory effects to common pathogens. The broth had thestrongest inhibitory activity to Erwinia carotovora in the 7 species of tested bacteria, withthe inhibition zone diameter 4.3mm, and Pyricularia grise was severely inhibited in the 6species of tested fugues, the inhibition rate is more than 99％.Stability tests showed that the fermentation time between 11～13d exhibited thestrongest inhibitory activity, and the inhibitory activity is lower with the increasing temperature, whereas the inhibitory activity were up to 52.804％when the broth weretreated at 100℃. Otherwise, the fermentation broth exhibited high activity under pH5.5～7.0, and it is likely sensitive to UV light.3. Antioxidant tests of different sources of Collybia radicata polysaccharides （drymycelium polysaccharide, fresh mycelium polysaccharide, extracelluar polysaccharide,cap polysaccharide, handle polysaccharide） and the fermentation broth showed that theyall had certain antioxidative activities.Various sources of polysaccharides had a strong DPPH- free radical scavengingeffect, which dry mycelium polysaccharide exhibited the strongest scavenging activitywith EC₅₀（DPPH.） value 0.394mg/mL, and the scavenging activities increased with theincreasing concentration. In contrast to the positive control vitamin C and vitamin E,scavenging effects of the different sources of polysaccharides were better than vitamin E,But worse than vitamin C.Reducing power test found that the value increased with the increase ofconcentration of polysaccharides. Extracelluar polysaccharide and dry myceliumpolysaccharide showed the greatest"reducing powers, with ECs0 （reducing power）,respectively 0.478mg/mL and 1.068mg/mL. But the reducing powers of various sourcesof polysaccharides were worse than the positive control vitamin C.Various sources of polysaccharides had a strong ferrous ion chelating capacity, andthe chelating capacity increased with the increasing concentration. Fresh myceliumpolysaccharide showed the greatest ferrous ion chelating capacity, with EC₅₀ （reducingpower） 0.126mg/mL. But the ferrous ion chelating capacities of various sources ofpolysaccharides were worse than the positive control EDTA.更多还原