Mechanism Of Stevioside Synthesis Regulation By Fulvic Acid And Its Enzymatic Modification

Excessive intake of sugar has caused serious health problems. Steviol glycosides (SGs) are natural, non-caloric high-intensity sweeteners that are extracted from leaves of Stevia rebaudiana Bertoni. Stevioside (ST) and rebaudioside A (RA) are the main constituents. With an additional glycosyl at position C13 of ST, RAhas a higher sweetness and much better edulcorant quality. The bitter aftertaste of ST due to its special structure restricts its application in the food industry for human direct consumption. The research on mono-glycosylation of the 13-O-β-sophorosyl moiety of ST to improve its sweetness and taste quality is of great significance. Fulvic acid (FA) is a natural plant growth regulator with the properties of promoting plant growth and improving crop quality. The study on regulation effect and mechanism of fulvic acid on Stevia growth and steviol glycosides synthesis could help to establish the theoretical and methodological basis for the development of new high quality sweeteners.Field experiments in Beijing, Anhui and Liaoning showed that foliar spray of FA solution (500 mg/L) could increase Stevia leaf yield and RA content by 9.2% and 14.3%, respectively. After the Box-Behnken design optimization of urea (8.9 kg/mu), humic acid (HA) (4.2 kg/mu) and FA (604.1 mg/L) applying amounts, the dry leaf yield and RA content significantly increased by 29.8% and 30%, compared to the control groups. The amount of urea applied was maintained at a relatively low level, which reduced about 80% of urea application compared to chemical fertilizations used in most plantations in China, which can reduce the cost of Stevia planting and diminish the environmental problems associated with conventional chemical fertilizers. The research on mechanism of improving RA content by FA showed that the UGT76G1 gene expression in FA treatment increased significantly which could lead to a quick RA synthesis in the leaves. The acceleration of ST transformed to RA through upregulating the UGT genes by FA treatment might be a promising solution to increase RA content in Stevia leaves.The community structure and variation of endophytic bacteria in Stevia leaves at different growth stages and their responses to FA treatment were investigated. Stevia growth stages strongly regulated the composition of endophytic community. The genera Agrobacterium and Erwinia dominated in seedling stage were apparently declined in the vegetable and initial flowering stages, while Sphingomonas and Methylobacterium increased in mature leaves at harvest time. Sphingomonas and Methylobacterium constituted an important part of the core endophytic community and were positively correlated with the stevioside content and UGT74G1 gene expression, respectively. FA treatment did not change the trend of endophytes variation along the growth stages, but it could accelerated the endophytes variation such as enrichment of potential benefit bacteria and decrease of plant pathogens, indicating FA could regulate the community structure and composition of endophytic bacteria. This study revealed the mechanism of increasing RA content by FA and the relationship between community structure of endophytic bacteria and steviol glycosides synthesis in Stevia, which could provide theoretical basis for improving Stevia leaf quality by regulating the endophytes community.In this study, a cyclodextrin glucanotransferase (CGTase) producing strain isolated from Stevia planting soil was identified as Paenibacillus sp.Y-6 by 16S rRNA gene sequencing. With starch as glycosyl donor, this CGTase can transform stevioside into a single specific product which is an isomer of rebaudioside A and identified as mono-glycosylated stevioside. Sensory evaluation showed the taste of stevioside was improved by generating mono-glycosylated stevioside, which possesses a sucrose-like taste and has sweetness and taste quality increased significantly by 35.4% and 36.2%, respectively. The parameters influencing CGTase production were optimized by response surface methodology. Compared to initial conditions, CGTase activity increased by 214.7% under optimum conditions of 3.9 g/L starch,17.9 g/L tryptone and 67.6 h of culture time, and the transglycosylation rate of stevioside was remarkably increased by 284.8%, reaching the highest productivity of mono-glycosylated stevioside (85.6%), and a high efficiency and low cost of stevioside modification method was preliminary established.