黄腐酸调控甜菊糖苷合成的机理及甜菊糖苷的酶法转化

发布时间：2018-06-17 10:40

  本文选题：甜菊糖 + 甜叶菊　； 参考：《中国农业大学》2016年博士论文

【摘要】：食糖的过量摄入已造成严重的健康问题。甜菊糖是从植物甜叶菊中提取出来的高甜度、无能量的天然甜味剂,其主要成分为甜菊糖苷(stevioside, ST)及甜菊糖A苷(rebaudioside A, RA).其中RA甜度高、口感良好。ST比RA在C-13位置少一个葡糖基,具有苦涩后味,限制了其在市场上的大范围推广,因此,通过转糖基酶在ST的C-13上连接一个葡糖基,改善甜菊糖苷甜度和味质的研究具有重要意义。黄腐酸是天然植物生长调节剂,具有刺激植物生长、改善作物品质的作用,研究黄腐酸对甜叶菊的生长及甜菊糖苷合成的调节作用及机理,有助于为开发新的优质甜味剂奠定理论和方法学基础。本研究通过在辽宁、北京和安徽的甜叶菊田间实验结果显示500 mg/L的黄腐酸处理可以使甜叶菊叶片产量至少提高9.2%,使RA含量至少提高14.3%。通过BBD响应面设计优化及田间实验验证,建立了FA施用浓度为604.1 mg/L,腐植酸和尿素施加量分别为4.2kg/亩和8.9kg/亩的施肥体系。与传统施肥体系相比,该体系可降低80%的尿素施用量,并提高叶片产量29.8%及RA含量30%,不但可以降低甜菊糖的生产成本,显著地改善了叶片品质,而且减少了化学肥料过度施用带来的环境问题。黄腐酸提高RA含量机理研究显示黄腐酸处理显著提高了甜叶菊叶片中RA合成过程中关键转糖基酶基因UGT76G1的表达量，从而促使ST转化为RA。初步阐明了黄腐酸处理促使甜叶菊体内定向加速转化ST为RA的过程,是提高RA含量的途径之一。进一步研究了甜叶菊不同生长阶段内生菌群落结构的组成与变化规律及其对黄腐酸处理的响应。结果表明不同生长阶段的甜叶菊叶片内生菌群落结构有显著差异,在苗期叶片中优势群落为Agrobacterium和Erwinia,在生长2个月和开花初期叶片中优势群落为Sphingomonas和Methylobacterium。随着生长时间的延长,Sphingomonas等相对丰度增加。相关性分析表明Sphingomonas和Methylobacterium等与叶片含糖量和转糖基酶基因的表达量具有显著的相关性。黄腐酸处理不改变内生菌群落结构随甜叶菊生长的变化趋势,但可以加速对植物生长代谢有益菌的相对富集,同时显著降低Erwinia等潜在致病菌的相对丰度。表明FA可以调节内生菌群落结构与组成,并能定向的提升有益菌、降低致病菌的相对丰度。本研究首次从内生菌角度揭示甜叶菊内生菌群落结构变化与甜菊糖合成的关系及黄腐酸调节糖苷合成的机理,为进一步探索通过调节内生菌群落提高甜叶菊品质提供了理论基础。从甜叶菊根际土壤中筛选到一株能产生较强环糊精葡萄糖基转移酶(CGTase)活性的菌株,16S rRNA基因序列分析鉴定为Paenibacillus sp. Y-6。该菌株产生的CGTase可以淀粉为糖基供体,在ST上转入一个葡糖基产生单一的转糖基产物单糖基-ST。该转化产物甜度比ST提高了35.40%,口感度提高了36.2%,其口感与蔗糖更相似。通过响应面法优化该菌株的培养条件,使其酶活提高了214.7%,达到69.2 U/mL。粗酶液对ST转化率提高了284.8%,达到了85.6%,单糖基-ST转化率是目前报道的最高值,初步建立了一种体外高效率低成本的甜菊糖苷味质改善方法。
[Abstract]:Excessive intake of sugar has caused serious health problems. Stevio is a high degree of sweetness extracted from plant sweet chrysanthemum, a natural sweetener without energy. Its main components are stevioside (ST) and stevioside A glycoside (rebaudioside A, RA). Among them, RA has high sweetness and a good taste.ST is less than RA in C-13. The bitter aftertaste limits its wide spread in the market. Therefore, it is of great significance to improve the sweetness and flavour of stevioside by connecting a glucoside on the C-13 of ST by glycosylase. The flavour acid is a natural plant growth regulator, which can stimulate plant growth and improve the quality of crops. The regulation and mechanism of the growth and the synthesis of stevioside are helpful to establish a theoretical and methodological basis for the development of new high quality sweeteners. The results of the field experiment in Liaoning, Beijing and Anhui showed that 500 mg/L of fulvic acid could increase the yield of leaves of Stevia by at least 9.2%, and at least 14 to increase the content of RA. .3%. through BBD response surface design optimization and field experiment proved that the application of FA with 604.1 mg/L, humic acid and urea applied to 4.2kg/ Mu and 8.9kg/ Mu respectively. Compared with the traditional fertilization system, the system can reduce the amount of urea application and increase leaf yield 29.8% and RA content 30%, not only can reduce the chrysanthemum. The production cost of sugar significantly improved the leaf quality and reduced the environmental problems caused by the excessive application of chemical fertilizers. The study on the mechanism of RA content increased by fulvic acid showed that the expression of the key glycosylase gene UGT76G1 in the RA synthesis process in the leaves of chrysanthemum was significantly improved, thus promoting the transformation of ST into RA.. The effect of fulvic acid treatment promoted the directional acceleration transformation of ST into RA in the body of Stevia, which was one of the ways to improve the content of RA. The composition and change rules of the endophytic community structure in different growth stages of chrysanthemum and the response to the treatment of fulvic acid were further studied. The results showed that the endophytic community knot of leaves of Stevia leaves at different growth stages. The dominant communities in the leaves of the seedling stage were Agrobacterium and Erwinia. The dominant communities in the leaves of the 2 months and the early flowering stages were Sphingomonas and Methylobacterium., and the relative abundance of Sphingomonas increased with the prolongation of the growth time. The correlation analysis showed that the sugar content of the leaves was Sphingomonas and Methylobacterium and so on. There is a significant correlation with the expression of transglycosylase genes. Fulvic acid treatment does not change the trend of the growth of endophyte community structure with the growth of chrysanthemum, but it can accelerate the relative enrichment of beneficial bacteria to plant growth and metabolism, and significantly reduce the relative abundance of Erwinia and other potential pathogens. It shows that FA can regulate the structure of endophytic bacteria community. It was the first time to reveal the relationship between the structure changes of the endophytic bacteria community and the synthesis of stevioside and the mechanism of the synthesis of glucoside to regulate the glucoside synthesis from the endophyte angle. This provides a theoretical basis for further exploring the improvement of the quality of the plant by regulating the endophyte community. A strain that produces a strong cyclodextrin glucotransferase (CGTase) activity is screened from the rhizosphere soil of the rhizosphere, and the sequence analysis of the 16S rRNA gene is identified as Paenibacillus sp. Y-6. and the CGTase produced by the strain can be a sugar based donor, and a single glucosyl product monosaccharide -ST., which is transferred to a glucosyl group on ST, is produced. The sweetness of the product was 35.40% higher than that of ST, and the taste was increased by 36.2%. Its taste was more similar to that of sucrose. By optimizing the culture condition of the strain, the enzyme activity was improved by 214.7%, and the conversion rate of ST was increased by 284.8% to 85.6%, and the conversion rate of monosaccharide -ST was the highest value reported at present. A high efficiency and low cost method for improving stevioside flavor in vitro was developed.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S566.9
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本文编号：2030750