本文選題：甜菊糖 + 甜葉菊　； 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：食糖的過量攝入已造成嚴(yán)重的健康問題。甜菊糖是從植物甜葉菊中提取出來的高甜度、無能量的天然甜味劑,其主要成分為甜菊糖苷(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位置少一個(gè)葡糖基,具有苦澀后味,限制了其在市場(chǎng)上的大范圍推廣,因此,通過轉(zhuǎn)糖基酶在ST的C-13上連接一個(gè)葡糖基,改善甜菊糖苷甜度和味質(zhì)的研究具有重要意義。黃腐酸是天然植物生長調(diào)節(jié)劑,具有刺激植物生長、改善作物品質(zhì)的作用,研究黃腐酸對(duì)甜葉菊的生長及甜菊糖苷合成的調(diào)節(jié)作用及機(jī)理,有助于為開發(fā)新的優(yōu)質(zhì)甜味劑奠定理論和方法學(xué)基礎(chǔ)。本研究通過在遼寧、北京和安徽的甜葉菊田間實(shí)驗(yàn)結(jié)果顯示500 mg/L的黃腐酸處理可以使甜葉菊葉片產(chǎn)量至少提高9.2%,使RA含量至少提高14.3%。通過BBD響應(yīng)面設(shè)計(jì)優(yōu)化及田間實(shí)驗(yàn)驗(yàn)證,建立了FA施用濃度為604.1 mg/L,腐植酸和尿素施加量分別為4.2kg/畝和8.9kg/畝的施肥體系。與傳統(tǒng)施肥體系相比,該體系可降低80%的尿素施用量,并提高葉片產(chǎn)量29.8%及RA含量30%,不但可以降低甜菊糖的生產(chǎn)成本,顯著地改善了葉片品質(zhì),而且減少了化學(xué)肥料過度施用帶來的環(huán)境問題。黃腐酸提高RA含量機(jī)理研究顯示黃腐酸處理顯著提高了甜葉菊葉片中RA合成過程中關(guān)鍵轉(zhuǎn)糖基酶基因UGT76G1的表達(dá)量，從而促使ST轉(zhuǎn)化為RA。初步闡明了黃腐酸處理促使甜葉菊體內(nèi)定向加速轉(zhuǎn)化ST為RA的過程,是提高RA含量的途徑之一。進(jìn)一步研究了甜葉菊不同生長階段內(nèi)生菌群落結(jié)構(gòu)的組成與變化規(guī)律及其對(duì)黃腐酸處理的響應(yīng)。結(jié)果表明不同生長階段的甜葉菊葉片內(nèi)生菌群落結(jié)構(gòu)有顯著差異,在苗期葉片中優(yōu)勢(shì)群落為Agrobacterium和Erwinia,在生長2個(gè)月和開花初期葉片中優(yōu)勢(shì)群落為Sphingomonas和Methylobacterium。隨著生長時(shí)間的延長,Sphingomonas等相對(duì)豐度增加。相關(guān)性分析表明Sphingomonas和Methylobacterium等與葉片含糖量和轉(zhuǎn)糖基酶基因的表達(dá)量具有顯著的相關(guān)性。黃腐酸處理不改變內(nèi)生菌群落結(jié)構(gòu)隨甜葉菊生長的變化趨勢(shì),但可以加速對(duì)植物生長代謝有益菌的相對(duì)富集,同時(shí)顯著降低Erwinia等潛在致病菌的相對(duì)豐度。表明FA可以調(diào)節(jié)內(nèi)生菌群落結(jié)構(gòu)與組成,并能定向的提升有益菌、降低致病菌的相對(duì)豐度。本研究首次從內(nèi)生菌角度揭示甜葉菊內(nèi)生菌群落結(jié)構(gòu)變化與甜菊糖合成的關(guān)系及黃腐酸調(diào)節(jié)糖苷合成的機(jī)理,為進(jìn)一步探索通過調(diào)節(jié)內(nèi)生菌群落提高甜葉菊品質(zhì)提供了理論基礎(chǔ)。從甜葉菊根際土壤中篩選到一株能產(chǎn)生較強(qiáng)環(huán)糊精葡萄糖基轉(zhuǎn)移酶(CGTase)活性的菌株,16S rRNA基因序列分析鑒定為Paenibacillus sp. Y-6。該菌株產(chǎn)生的CGTase可以淀粉為糖基供體,在ST上轉(zhuǎn)入一個(gè)葡糖基產(chǎn)生單一的轉(zhuǎn)糖基產(chǎn)物單糖基-ST。該轉(zhuǎn)化產(chǎn)物甜度比ST提高了35.40%,口感度提高了36.2%,其口感與蔗糖更相似。通過響應(yīng)面法優(yōu)化該菌株的培養(yǎng)條件,使其酶活提高了214.7%,達(dá)到69.2 U/mL。粗酶液對(duì)ST轉(zhuǎn)化率提高了284.8%,達(dá)到了85.6%,單糖基-ST轉(zhuǎn)化率是目前報(bào)道的最高值,初步建立了一種體外高效率低成本的甜菊糖苷味質(zhì)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S566.9
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本文編號(hào)：2030750