【作者】 蔡文韬；

【导师】 夏延斌；

【作者基本信息】 湖南农业大学 ， 园艺产品采后科学与技术， 2013， 博士

【摘要】 果蔬采后病害是影响其保鲜和贮藏的主要因素之一,目前使用化学类制剂减少病害仍是主要措施,但剂量不适宜造成残留污染；连续使用易诱发病原菌的抗药性；造成环境污染；危害人们的健康等。而健康的植物体内存在大量的内生菌,作为生防菌将是一个新的资源库,在寻找新的控制果蔬采后病害方面,具有良好的应用前景。本课题以健康辣椒为研究对象,从中筛选出对辣椒多种病原菌具有拮抗作用的内生菌,并从形态学特征、生理生化反应、分子生物学方面进行了生物学鉴定,对抑菌效果好的菌株进行了培养条件优化、抗菌活性物质分离、纯化与鉴定。主要结论如下：1研究了辣椒植株不同部位和品种内生菌的分布多样性。从辣椒植株根、茎、叶和果肉中共分离出27株内生细菌,7株内生真菌,从柿子椒、尖椒(绿色)、朝天椒(红色)、线椒不同品种共分离12株内生细菌,3株内生真菌,从果肉中分离到4株具有明显拮抗作用的内生细菌；其中一株试验号为strain-L抑菌效果最好,对链格孢菌、辣椒炭疽病菌、辣椒疫霉菌抑菌直径分别为27.71mm、24.8mm、19.45mm。另一株strain-39对链格孢菌、辣椒疫霉菌及细菌性软腐菌抑制直径分别为10.45mm、6.9mm、17.82mm。2strain-L和strain-39菌株鉴定。对strain-L和strain-39内生菌株进行分子生物学鉴定。strain-L序列与解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的序列相似性达到99%,可确定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens), strain-L的16SrDNA长度1452bp; strain-39的18SrRNA序列与粘质酵母菌序列相同,可确定为黏质红酵母(Rhodotorula mucilaginosa), strain-39的18rRNA长度为539bp。3strain-39抗菌特性初步研究对strain-39发酵滤液有机溶剂萃取液和乙醇沉淀提取液抑菌效果进行了研究,结果表明：75%乙醇沉淀物和乙酸乙酯提取物对茄科链格孢菌产孢、萌发和菌丝的生长等方面都具有较好抑菌效果,75%乙醇沉淀物EC5o为16.3531mg.L-1,乙酸乙酯提取物EC50为11.8003mg.L-1,将其应用于食品(辣椒、番茄)防腐上也有一定的效果,乙酸乙酯浓度为100mg/mL时,辣椒腐烂率下降了51.87%,番茄腐烂率下降了58.47%,75%乙醇沉淀物为100mg/mL时,辣椒腐烂率下降了47.50%,番茄腐烂率下降了50.47%。strain-39菌液75%乙醇沉淀物和乙酸乙酯提取物经过不同酸碱和温度处理后,抑菌效果存在差异,紫外诱变处理后对链格孢菌抑菌效果基本没有影响。由此分析,strain-39抑菌活性是多种成分共同作用结果。4strain-L生物学特性研究。将该菌在不同条件下培养,结果显示菌株在pH5.2-8.0范围内能较好的生长,最适生长pH为7.2;生长温度范围为25℃-30℃,最适生长温度为28℃,菌株同时具有较强的渗透压适应能力,能够利用大多数的碳源和有机氮源,以及少数的无机氮源,说明菌株的适应性较强。5strain-L发酵液对辣椒采后保鲜效果研究。利用从柿子椒果肉里筛选出的strain-L发酵滤液对不同辣椒品种(海青椒、红尖椒)贮藏效果进行初步研究。体内体外试验结果表明不同浓度strain-L发酵液对链格孢菌具有较好的抑菌效果。损伤接种25℃下贮藏7d,strain-L发酵液对海青椒和红尖椒都具有明显的防腐效果,与无菌水相比,海青椒腐烂率下降了51.87%,红尖椒下降了58.54%。非损伤接种9℃下贮藏24d, strain-L发酵液对不同辣椒品种保鲜效果存在差异,在整个贮藏期间,strain-L发酵液处理可降低辣椒采后腐烂率,明显优于无菌水组。贮藏24d后,青椒发酵液处理和保鲜液处理的腐烂率分别为37.5%,32.50%,与无菌水组相比,分别降低了35%和40%。红尖椒的发酵液处理和保鲜液处理的腐烂率分别为29.63%,24.14%,与无菌水组相比降低了34.19%,28.70%,不同品种间,发酵液处理组和保鲜液处理组的腐烂程度不同,且在整个贮藏期间,strain-L发酵液延缓了果实内还原糖含量、可溶性固形物、可溶性蛋白、Vc的降低,保持了辣椒较稳定的色泽和质构。Strain-L发酵液在保持辣椒商品价值、感官品质以及营养价值方面获得了较好的贮藏效果。6strain-L的发酵培养条件优化。利用均匀设训(Uniform Design, UD)与支持向量回归(Support Vector Regression, SVR)结合的方法(UD-SVR),得到最优的培养基配方：淀粉0.5%、葡萄糖2%、酵母提取物0.4%、CaCL21.3%、KH2PO40.1%,最优培养条件：温度30℃、时间36h、接种量1%和pH值7.0。在此条件下得到的发酵液稀释10倍抑菌活性达到57.39%,比基准试验高出20.53%。7strain-L菌株抑菌活性组分提纯与分离。strain-L1菌株发酵液经正丁醇萃取、硅胶柱层析、制备液相色谱分离,共分离出4种具有较强抑菌活性组分,分别为5、16、17、18号峰,对链格孢菌孢子萌发抑制率分别为：20.73%、27.43%、13.76%、28.5%,分析型RP-HPLC纯度分析结果表明：4种组分为相对单一峰,采用6530型-四级杆飞行质谱-高效液相联用仪(UHPLC-Q-TOF)对5,16,17,18四个组分进行结构鉴定,分子量分别为253Da、366Da、1131Da、384.2Da,解析出可能分子式为C10H23NO6、C24H30O3/C17H30N6OS、C55H87O24、C24H32O4。对抑菌活性最强的组分17进行深入研究,结合质谱、红外、1H-NMR、13C-NMR,确定为21β-巴豆酰基-22α-乙酰基原七叶皂苷配基-3β-O-[β-D-吡喃葡萄糖基(1-2)][β-D-吡喃葡萄糖基(1-4)]-β-D-吡喃葡萄糖醛酸。更多还原

【Abstract】 Postharvest diseases of fruit and vegetable is one of the main factors affecting on preservation and storage, the use of chemical agent is still the main measures recently, but unfittable does will cause residual pollution; induce drug resistance of pathogenic bacteria easily; Cause environmental pollution; Harm people’s health and so on. the healthy plants presented arge amounts of endophytes which will be a new repository as bio-control bacteria, it has a good application prospect, in terms of searching for new controlling postharvest diseases of fruits and vegetables. Take chilli as object, it was selected from healthy plants of pepper which have antagonistic effect on alternaria alternate and Colletotrichum capsici, it was identified by the morphological characteristics, physiological and biochemical reactions, and molecular biology. It was studied on the culture conditions optimization, antimicrobial active, substances separation basis of good strains of bacteria inhibiting effect of chili.1the distribution diversity of endogenous bacteria in different parts of chilli different kinds of chilli pulp.27strains endophytic bacteria and7strains fungis were isolated from stems, roots, leaves and fruit of capsicum from pepper plant,12strains were isolated from different types of pepper.3strain of endophytic fungi,5of them showed antifungal effect against the pathogens, the strain-L in bell pepper was the best antagonistic to Alternaria alternate, Colletotrichum capsici and Phytophthora capsici, the bacteriostatic diameter was27.71mm,24.8mm,19.45mm respectly. The strain-39was good antagonistic to Alternaria alternate, Colletotrichum capsici, the bacteriostatic diameter was10.45mm、6.9mm、17.82mm respectly.2the identification of strain-L and strain-39. Analysis of the characteristics of morphology, Physiology, bioehemistry and the16SrDNA, strain L was identified as a strain of Bacillus subtilis, which had99%sequence similarity with B. subtilis strain-39was identified as a strain of Rhodotorula mucilaginosa, which had99%sequence similarity with Uncultured basidiomycota genomic. strain-L and strain-39have the16SrDNA length of1452bp and539bp.3Antibacterial mechanism research ethanol sediment and absolute ethyl alcohol of strain-39fermentation was studied, The results show that75%ethanol sediment and ethyl acetate extract produced from strain-39had a good antibacterial effect on Alternaria alternata mycelial growth, spores bearing and germination, the EC50of the ethyl acetate extract is16.3531mg/L-1, the EC50of75%ethanol sediment is11.8003mg/L-1, it also had a significant inhibitory effect on the food (pepper, tomato), when the concentration of ethyl acetate extract was100mg/mL, the decay rates of peppers and tomats were reduced by51.897%and58.47%, the concentration of75%ethanol sediment was100mg/mL, the decay rates of peppers and tomats were reduced by47.51%and50.47%. Growth inhibition rate had significant difference between75%ethanol sediment and ethyl acetate extract at various pH (2-12) and different temperature treatment, it had no diference at ultraviolet treatment. Overall, this study indicates that the strain-39active substance is composed of various components.4the research on biological characteristics, the result showed that the pH value and temperature has obviously effect on the growth of the strain, strain can growth better in the range of the pH5.2-pH8.0and25℃-30℃, the optimal growth of pH and is7.2and28℃respectly, which have strong osmotic pressure at the same time, strain-L can be able to use all of the organic nitrogen source, carbon source and few unorganic nitrogen source, which explain that the strain have strong adaptability without strict requirements.5The effective impact to preserve long pepper of different varieties treated with fermented filtrated liquor of strain-L. the results of invivo and invitro tests indicated that different concentrations strain-L fermentation medium had a good antibacterial effect on A.alternate, Through the damage inoculation, strain-L fermentation medium has obvious antibacterial effect on the green pepper and red chilli, the decay rates of green peppers were declined by51.87%. compared with sterile water, decay rate of the red chilli were declined by58.54%compared with sterile water, the preservation effect on strain-L fermentation medium on different varieties of pepper had significant difference during24d storage period by noninvasive inoculation, the results showed that the strain-L fermented liquid treatment can obviously decrease the decay rate of chili postharvest in the storage period, was obviously superior to the sterile water group. After24d, the decay rate of green pepper treated with fermentation medium and preservation liquid were37.5%, 32.50%respectly, decreased by35%and40%respectively compared to sterile water group, the level of decay rates was different with fermentation medium treatment group and preservation liquid treatment between different varieties. In the storage period, the strain-L delayed the reducing sugar content, inhibit the loss of soluble solid content and soluble protein, maintain the stable color and quality structure. Strain-L fermentation liquor obtained the good storage effect in keeping the chili good commodity value, sensory quality and nutritional value.6optimization of fermentation culture conditions. Object as strain L, a novel experimental design and analysis approach named uniform design and support vector regression (UD-SVR) was proposed to optimize alternative culture medium components and fermenter conditions. The optimal formula considering9factors through3rounds of mixed level experiments was starch0.5%, glucose2%, yeast extract0.4%, CaCl21.3%, KH2PO40.1%,30℃,36h, inoculation amountl%and pH7.0. The inhibitory activity reached57.39%of10fold diluted fermented liquid,20.53%higher than the benchmark formulation of the single factor. UD-SVR is more efficient than the referencing models and has potentials to be widely used for experimental design and analysis of the prescription optimization.7purification and separation of components of fermented liquid of strain L. It was extracted by n-butyl alcohol and silica gel column chromatography and liquid chromatograph separation of the strain-L, a total of four bacteria inhibitory active component has been isolated, the spores germination inhibition rate to A alternate respectively were part5(3.213min)20.73%、16(22.618min) components27.43%、17(23.783min) group13.76%,18(27.210min) group of28.5%, It was identify the molecular weight and constitutional formula of four components by the HPLC/ESI-TOF/MS tandem mass spectrometry, respectively, and the molecular were253Da、366Da、1131Da、384.2Da, C,oH23NO6、C24H30O3/C17H30N6OS、C55H87O24、 C24H32O4. respectly. research on inhibitory activity of the strongest component17deeply, combined with mass spectrometry, IR and1H-NMR,13C-NMR, identified as aesicn compounds.更多还原