生防酵母（Candida diversa）形态变化及金属离子提高其发酵生物量和生防效力的研究

发布时间：2018-12-14 10:00

【摘要】：新鲜水果产品是人们必不可少的营养来源,在国民经济中起着举足轻重的作用。然而由于病原菌侵染水果,引起腐烂造成巨大的经济损失。目前水果采后病害的主要防治措施仍是使用化学农药,但化学农药对人类健康、生态环境和食品安全产生的不利影响正越发受到人们的关注。近年来,使用生物拮抗菌防治水果采后病害是替代化学农药防治的安全有效措施之一。本课题以酵母拮抗菌(Candida diversa)为研究对象,酵母拮抗菌在形态上的变化(从酵母单细胞形态到生物膜形态)是基于酵母菌的生物防治系统中一种新的作用模式。探索出酵母拮抗菌产生形态变化的条件,酵母单细胞形态和生物膜形态的C.diversa对热逆境和氧化逆境的耐受性以及对苹果、猕猴桃灰霉病防治效果的影响。同时为获得较大生物量的拮抗酵母菌应用到农业生产中,本课题也进行拮抗酵母菌发酵培养基优化。通过响应面设计实验探索出发酵培养基三种关键金属离子(Mg2+、Fe2+、Zn2+)对拮抗酵母菌生物量和存活率影响的研究,对比不同金属离子浓度对发酵中的拮抗酵母菌的存活率和生理生化特性的影响。主要结论如下:(1)酵母拮抗菌产生形态变化的条件:当拮抗酵母菌C.diversa从琼脂含量为2%的YPD培养基转移到琼脂含量为0.3%的YPD培养基时,其形态由酵母形态转变为生物膜形态。(2)与酵母单细胞形态相比,生物膜形态的C.diversa对热逆境和氧化逆境的耐受性以及对苹果、猕猴桃灰霉病的防治效果有着显著地增强。(3)拮抗酵母菌发酵培养基优化:通过单因素试验和响应面试验,对拮抗酵母菌发酵培养基金属离子含量进行优化。培养基关键离子含量最佳条件为:Mg2+为1511.13 mg/L,Fe2+浓度为2.16 mg/L,Zn2+浓度为26.77 mg/L,有效生物量为5.28 g。(4)实验表明初始培养基金属离子浓度对C.diversa的存活率、生物量积累和生理生化有着十分重要的影响。与MM(最小矿物培养基)和Lack(Mg2+、Fe2+和Zn2+三种离子缺失)实验组相比,Best(响应面优化Mg2+、Fe2+和Zn2+三种离子浓度)实验组有着较高的生物量、存活率和生防效力,同时其抗氧化酶活性较高并且蛋白损伤和脂质氧化程度较低。经优化的培养基可以显著提高C.diversa的抗逆能力和生防效力。研究结果表明,当YPD培养基琼脂含量由2%变成0.3%时,拮抗酵母菌C.diversa酵母形态可以转变为生物膜形态,生物膜形态的C.diversa对逆境的耐受性以及对苹果灰霉病的防治效果有着显著增强,Mg2+、Fe2+、Zn2+对拮抗酵母菌发酵生物量积累有着积极的作用,提高了发酵过程中酵母菌的存活率,增强了其超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性,降低丙二醛(MDA)和羰基化蛋白含量。本研究提供了新的关于酵母菌拮抗菌形态变化对其非生物胁迫、生物防治效果以及金属离子对发酵生物量积累影响的研究结果,这对提高拮抗菌的应用价值是非常必要的。
[Abstract]:Fresh fruit is an essential source of nutrition and plays an important role in the national economy. However, due to the infection of fruit by pathogenic bacteria, the rot caused huge economic losses. At present, the main control measures of fruit postharvest diseases are still the use of chemical pesticides, but the adverse effects of chemical pesticides on human health, ecological environment and food safety are getting more and more attention. In recent years, the use of bioantagonistic bacteria to control postharvest diseases of fruits is one of the safe and effective measures to replace chemical pesticides. In this study, yeast antagonistic bacteria (Candida diversa) were studied. The morphological changes of yeast antagonists (from yeast single cell morphology to biofilm morphology) are a new mode of biological control system based on yeast. The conditions for morphological changes of yeast antagonistic bacteria, the effects of single cell morphology and biofilm morphology of yeast C.diversa on thermal and oxidative stress, and on the control effect of apple and kiwifruit grey mold were explored. At the same time, in order to obtain large biomass of antagonistic yeast used in agricultural production, this subject also optimized the fermentation medium of antagonistic yeast. The effects of three key metal ions (Mg2, Fe2, Zn2) on the biomass and survival rate of antagonistic yeast were investigated by response surface design. The effects of different concentrations of metal ions on the survival rate and physiological and biochemical characteristics of antagonistic yeast in fermentation were compared. The main conclusions are as follows: (1) the condition of morphological change of yeast antagonistic bacteria: when the antagonistic yeast C.diversa was transferred from YPD medium containing 2% Agar to YPD medium with 0.3% Agar content, (2) compared with the single cell form of yeast, the C.diversa of biofilm form is resistant to heat stress and oxidative stress, and to apple. The control effect of Actinidia cinerea was significantly enhanced. (3) Optimization of fermentation medium for antagonistic yeast: through single factor test and response surface test, the content of metal ions in the fermentation medium of antagonistic yeast was optimized. The optimum conditions for the content of key ions in culture medium are as follows: Mg2 is 1511.13 mg/L,Fe2, concentration is 2.16 mg/L,Zn2, concentration is 26.77 mg/L,. The effective biomass was 5.28 g. (4) the experimental results showed that the concentration of metal ions in the initial culture medium had a very important effect on the survival rate, biomass accumulation, physiological and biochemical characteristics of C.diversa. Compared with MM (minimum mineral medium) and Lack (Mg2, Fe2 and Zn2) groups, Best (response surface optimization of Mg2, Fe2 and Zn2 ion concentrations had higher biomass, survival rate and biocontrol efficacy. At the same time, its antioxidant enzyme activity is higher and the degree of protein damage and lipid oxidation is lower. The optimized medium can significantly improve the stress resistance and biocontrol effect of C.diversa. The results showed that when the Agar content of YPD medium changed from 2% to 0.3%, the yeast form of antagonistic yeast C.diversa could be transformed into biofilm form. The tolerance of biofilm form C.diversa to stress and the control effect to apple grey mold were significantly enhanced. Mg2, Fe2 and Zn2 had positive effects on antagonizing the accumulation of fermentation biomass of yeast. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were increased, and the contents of malondialdehyde (MDA) and carbonylated protein were decreased. This study provides new research results on the effects of antagonistic morphological changes of yeast on abiotic stress, biological control and the accumulation of metal ions on fermentation biomass, which is very necessary to improve the application value of antagonistic bacteria.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476.1

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