The need for medicinal and aromatic plants for industrial uses creates an opportunity for farmers to produce alternative crops. Stevia rebaudiana Bertoni, a perennial shrub originating from Paraguay, is of increasing interest as a source of zero-calorie natural sweeteners: the steviol glycosides (SVglys). The aim of this study was to investigate the relevance of nitrogen (N) supply for leaf yield and for SVgly concentrations in leaves, which are the two major components of S. rebaudiana productivity. In this regard, the relationship between leaf N concentration, CO2 assimilation, leaf production and SVgly accumulation was investigated. The experiments were conducted consecutively in growth-chamber (CC: controlled conditions), in greenhouse (SCC: semi-controlled conditions) and in field conditions (FC) on two genotypes. In CC and SCC, three levels of N fertilization were applied. Plants were grown on four locations in the FC experiment. Both N supply (CC and SCC) and location (FC) had a significant effect on N content in leaves. When light was not limiting (SCC and FC) N content in leaves was positively correlated with CO2 assimilation rate and biomass accumulation. Irrespective of the growth conditions, N content in leaves was negatively correlated with SVgly content. However, increased SVgly content was correlated with a decreased ratio of rebaudioside A over stevioside. The evidence that the increased SVgly accumulation compensates for the negative effect on biomass production suggests that adequate SVgly productivity per plant may be achieved with relatively low fertilization.

Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub native to Paraguay whose leaves accumulate sweetening compounds of high value: steviol glycosides (SVglys). These diterpenoids differ in the number (1-4) and the nature of sugar units bound to a steviol skeleton: mainly glucose but also rhamnose and xylose. The two main SVglys are stevioside (ST; 3 glucose units) and rebaudioside A (RA, 4 glucose units). Along with an evaluation of genotypic variability for SVglys content and composition, this study investigates the winter survival and the effect of perennial cultivation on S. rebaudiana genotypes through a 2-year experiment conducted on two locations. The first year, a population of 96 genotypes was examined in a nursery field (site A) and a subset of five genotypes was grown in a field trial (site B). The winter surviving genotypes (16 on site A and 5 on site B) were studied the following year, alongside new plantings of the same lines (8 on site A and 5 on site B). The genotypes showed a high variability in SVgly content (from 45.7 to 186.2 mg g -1 DM) and composition along four distinct clusters. There was no correlation between total SVglys content and composition. Genotypes without RA had no other SVglys with more than three conjugated sugars. SVgly content varied among environments and generally increased between 1-and 2-year-old plants, while SVgly composition remained stable. This study showed that breeding for winter survival in temperate conditions would allow an increase in SVgly productivity in a semi-perennial stevia production.

Aqueous extracts of Stevia rebaudiana leaves have been approved since 2008 by the Joint Expert Committee for Food Additives as sugar substitutes in many food and beverages in Western and Far East Asian countries. The compounds responsible for the natural sweetness of Stevia leaves include a diversity of diterpenoid glycosides derived from a steviol skeleton. These steviol glycosides also exhibit a low calorific value as well as promising therapeutic applications, particularly for the treatment of sugar metabolism disturbances. In this work, centrifugal partition chromatography is proposed as an efficient technical alternative to purify steviol glycosides from crude aqueous extracts of Stevia leaves on a multigram scale. Two different commercial instruments, including an ASCPC250® and a FCPE300® made of columns containing 1890 and 231 twin-cells, respectively, were evaluated and compared. All experiments were performed with a polar biphasic solvent system composed of ethyl acetate, n-butanol, and water in a gradient elution mode. When using the 1890 partition cell centrifugal partition chromatography column of 250 mL, 42 mg of stevioside, 68 mg of dulcoside A, and 172 mg of rebaudioside A, three major constituents of the initial extract were obtained from 1 g of the initial mixture at purities of 81%, 83%, and 99%, respectively. The productivity was further improved by intensifying the procedure on the 231 partition cell centrifugal partition chromatography column of 303 mL with the sample mass loading increased up to 5 g, resulting in the recovery of 1.2 g of stevioside, 100 mg of dulcoside A, and 1.1 g of rebaudioside A at purities of 79%, 62%, and 98%, respectively. The structures of the isolated compounds were validated by HPLC-UV, ESI-MS, (1)H, and (13)C NMR analyses. Altogether, the results demonstrate that the column design (i.e., the partition cell number) is an important aspect to be considered for a larger scale centrifugal partition chromatography isolation of Stevia-derived natural sweeteners.