The aim of this study was to learn the interactions among the endophytic bacteria, the plant growth, the foliar spray of fulvic acid, and the accumulation of steviol glycosides in the leaves of Stevia rebaudiana. Metagenomic DNA was extracted from the Stevia leaves at different growth stages with or without the fulvic acid treatment; and the diversity of endophytic bacteria in Stevia leaves was estimated by pyrosequencing of 16S rRNA genes. As results, Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were found to be the dominant phyla despite the growth stages and fulvic acid application. Stevia growth stages strongly regulated composition of endophytic community. The genera Agrobacterium (12.3%) and Erwinia (7.2%) 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, which showed that the mature leaves of Stevia preferred to accumulate some certain endophytic bacteria. 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; while Erwinia, Agrobacterium, and Bacillus were negatively correlated with the stevioside accumulation. Fulvic acid treatment accelerated the variation of endophytes along the growth stages and increased the steviol glycosides content. This is the first study to reveal the community composition of endophytic bacteria in the Stevia leaves, to evidence the strong effects of growth stage and fulvic acid application on the endophytes of Stevia, and to demonstrate the correlation between the endophytic bacteria and the steviol glycosides accumulation.

The transplantation of tissue-engineered scaffolds with stem cells is a promising therapeutic approach for bone defect repair. To improve the therapeutic efficacy of this approach, in this study, a novel biofunctional live tissue-engineered bone-like graft was designed and constructed using a fibrin scaffold loaded with TG2 gene-modified ectomesenchymal stem cells (TG2-EMSCs) derived from nasal respiratory mucosa for bone defect repair. Autocalcification of the cell-free fibrin gel in osteogenic medium with additional alkaline phosphatase (ALP) and the osteogenic differentiation of TG2-EMSCs on the fibrin scaffold were assessed in vitro. The results indicated that the cell-free fibrin gel could autocalcify in the osteogenic medium with ALP and that the overexpression of TG2 by TG2-EMSCs could promote the osteogenic differentiation of these stem cells in the fibrin scaffold. Moreover, TG2 could enhance the deposition of extracellular matrix proteins in the fibrin scaffold, followed by calcification of the bone matrix in vitro. After transplantation into critical-sized cranial defects in rats, the functional tissue-engineered bone-like grafts improved bone regeneration. These results indicate that this tissue-engineered bone-like graft could improve the process of bone defect repair.

Stevioside is a non-caloric, natural, high-intensity sweetener. However, the bitter aftertaste of stevioside restricts its utilization for human consumption and limits its application in the food industry. In this study, a high efficiency enzymatic modification system was investigated to improve stevioside taste quality. A cyclodextrin glucanotransferase (CGTase) producing strain Paenibacillus sp. CGMCC 5316 was isolated from Stevia planting soil. 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. The taste of stevioside is improved noticeably by generating mono-glycosylated stevioside, which possesses a sucrose-like taste and has sweetness increased significantly by 35.4%. Next, the parameters influencing CGTase production were optimized. 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 85.6%. This CGTase modification system provides a promising solution for improving the sweetness and taste quality of stevioside. The efficiency of CGTase transformation can be greatly increased by optimizing the culture conditions of Paenibacillus sp. CGMCC 5316.

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