HI-HPTLC-UV/Vis/FLD-HESI-HRMS and bioprofiling of steviol glycosides, steviol, and isosteviol in Stevia leaves and foods.
ABSTRACT: Food products and botanicals on the global market need to be investigated in a more comprehensive way to detect effects, falsifications or adulterations. This is especially true for such ones containing Stevia leaves, Stevia extracts, or steviol glycosides. A multi-imaging profiling was developed exploiting hydrophilic interaction liquid chromatography (HILIC). A minimalistic sample preparation, different mixtures of acetonitrile and water/buffer on the silica gel phase as well as derivatization reagents and optional hyphenation with high-resolution mass spectrometry were exploited. The hydrophilic interaction high-performance thin-layer chromatography (HI-HPTLC) development took 10 min for 48 analyses. It was used to screen Stevia leaf extracts and 20 different food products. For the first time, the biological and biochemical profiling of Stevia leaf products by HI-HPTLC-UV/Vis/FLD-assay pointed to 19 different bioactive compound bands found in the more natural multicomponent Stevia leaf extracts, whereas most of these activities were not existent for the steviol glycosides. The chemically isolated, purified, and EU-regulated steviol glycosides ease risk assessment and food product development. However, multipotent botanicals may have subtle impact on homeostasis via several metabolic pathways, providing benefits for the consumer's health. Analyzed side by side by means of the effect-directed profiling, their individual activity profiles were visualized as image and individual substances of importance were pointed out. Multi-imaging (comprehensive detection) plus non-targeted bioprofiling (focus on known and unknown bioactivity) allows for a fast detection of questionable product changes that occur along the global food chain and are particularly related to food safety. Graphical abstract.
Project description:Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1-2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1-2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1-2) glucosylation in two distinct orientations as well as β (1-6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1-2) glucosylation, eliminate β (1-6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.
Project description:Stevia (Stevia rebaudiana Bertoni) is well known for its very sweet steviol glycosides (SGs) consisting of a common tetracyclic diterpenoid steviol backbone and a variable glycone. Steviol glycosides are 150-300 times sweeter than sucrose and are used as natural zero-calorie sweeteners. However, the most promising compounds are biosynthesized in small amounts. Based on Illumina, PacBio, and Hi-C sequencing, we constructed a chromosome-level assembly of Stevia covering 1416 Mb with a contig N50 value of 616.85 kb and a scaffold N50 value of 106.55 Mb. More than four-fifths of the Stevia genome consisted of repetitive elements. We annotated 44,143 high-confidence protein-coding genes in the high-quality genome. Genome evolution analysis suggested that Stevia and sunflower diverged ~29.4 million years ago (Mya), shortly after the whole-genome duplication (WGD) event (WGD-2, ~32.1 Mya) that occurred in their common ancestor. Comparative genomic analysis revealed that the expanded genes in Stevia were mainly enriched for biosynthesis of specialized metabolites, especially biosynthesis of terpenoid backbones, and for further oxidation and glycosylation of these compounds. We further identified all candidate genes involved in SG biosynthesis. Collectively, our current findings on the Stevia reference genome will be very helpful for dissecting the evolutionary history of Stevia and for discovering novel genes contributing to SG biosynthesis and other important agronomic traits in future breeding programs.
Project description:Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP-glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3-?-d-glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild-type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S-transferase-tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.
Project description:<i>Stevia rebaudiana</i> Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a β-glucosidase, <i>Mt</i>Bgl3a, and a β-galactosidase, <i>Tt</i>bGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by <i>Mt</i>Bgl3a and <i>Tt</i>bGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for <i>Tt</i>bGal1- and <i>Mt</i>Bgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.
Project description:Steviol glycosides were subjected to bacteria present in a soil sample collected from a Stevia plantation in Paraguay. During the incubation experiments, next to the aglycon steviol, steviol degradation products were also formed. X-ray analysis and NMR methods in combination with chemical synthesis and GIAO NMR calculations were used to fully characterize the structure of these compounds as a tricyclic ketone and the corresponding reduced form. They were nicknamed <i>monicanone</i> and <i>monicanol</i>. The latter has the (<i>S</i>)-configuration at the alcohol site.
Project description:Governments are creating regulations for consumers to reduce their sugar intake, prompting companies to increase the ratio of artificial sweeteners in their products. However, there is evidence of some deleterious effects ascribed to the aforementioned synthetic agents and therefore consumers and food manufacturers have turned their attention to natural dietary sweeteners, such as stevia, to meet their sweetening needs. Stevia is generally considered safe; however, emerging scientific evidence has implicated the agent in gut microbial imbalance. In general, regulation of microbial behavior is known to depend highly on signaling molecules via quorum sensing (QS) pathways. This is also true for the gut microbial community. We, therefore, evaluated the possible role of these stevia-based natural sweeteners on this bacterial communication pathway. The use of a commercial stevia herbal supplement resulted in an inhibitory effect on bacterial communication, with no observable bactericidal effect. Purified stevia extracts, including stevioside, rebaudioside A (Reb A), and steviol revealed a molecular interaction, and possible interruption of Gram-negative bacterial communication, via either the LasR or RhlR receptor. Our in-silico analyses suggest a competitive-type inhibitory role for steviol, while Reb A and stevioside are likely to inhibit LasR-mediated QS in a non-competitive manner. These results suggest the need for further safety studies on the agents.
Project description:Steviol glycosides (SGs), such as stevioside and rebaudioside A, are natural, non-caloric sweet-tasting organic molecules, present in extracts of the scrub plant Stevia rebaudiana, which are widely used as sweeteners in consumer foods and beverages. TRPM5 is a Ca<sup>2+</sup>-activated cation channel expressed in type II taste receptor cells and pancreatic β-cells. Here we show that stevioside, rebaudioside A and their aglycon steviol potentiate the activity of TRPM5. We find that SGs potentiate perception of bitter, sweet and umami taste, and enhance glucose-induced insulin secretion in a Trpm5-dependent manner. Daily consumption of stevioside prevents development of high-fat-diet-induced diabetic hyperglycaemia in wild-type mice, but not in Trpm5<sup>-/-</sup> mice. These results elucidate a molecular mechanism of action of SGs and identify TRPM5 as a potential target to prevent and treat type 2 diabetes.
Project description:BACKGROUND:miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents. RESULTS:The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents. CONCLUSION:In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.
Project description:<h4>Aims</h4>Stevia rebaudiana Bertoni leaf extracts have gained increasing attention for their potential protection against type 2 diabetes. In this study, we have evaluated the possible beneficial effects of Stevia rebaudiana leaf extracts on beta-cells exposed to lipotoxicity and explored some of the possible mechanisms involved.<h4>Methods</h4>Extracts, deriving from six different chemotypes (ST1 to ST6), were characterized in terms of steviol glycosides, total phenols, flavonoids, and antioxidant activity. INS-1E beta cells and human pancreatic islets were incubated 24 h with 0.5 mM palmitate with or without varying concentrations of extracts. Beta-cell/islet cell features were analyzed by MTT assay, activated caspase 3/7 measurement, and/or nucleosome quantification. In addition, the proteome of INS-1E cells was assessed by bi-dimensional electrophoresis (2-DE).<h4>Results</h4>The extracts differed in terms of antioxidant activity and stevioside content. As expected, 24 h exposure to palmitate resulted in a significant decrease of INS-1E cell metabolic activity, which was counteracted by all the Stevia extracts at 200 μg/ml. However, varying stevioside only concentrations were not able to protect palmitate-exposed cells. ST3 extract was also tested with human islets, showing an anti-apoptotic effect. Proteome analysis showed several changes in INS-1E beta-cells exposed to ST3, mainly at the endoplasmic reticulum and mitochondrial levels.<h4>Conclusions</h4>Stevia rebaudiana leaf extracts have beneficial effects on beta cells exposed to lipotoxicity; this effect does not seem to be mediated by stevioside alone (suggesting a major role of the leaf phytocomplex as a whole) and might be due to actions on the endoplasmic reticulum and the mitochondrion.
Project description:BACKGROUND:Stevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking. RESULTS:Here, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42-54% and 67-88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants. CONCLUSION:This study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.