Daminozide Enhances Vigor and Steviol Glycoside Yield of Stevia (Stevia rebaudiana Bert.) Propagated in the Temporary Immersion Bioreactor

Stevia (Stevia rebaudiana Bertoni) contains sweet compound widely used as natural sweetener, steviol glycoside (SG). SG is a diterpenoid secondary metabolite synthesized from ent-kaurenoic acid, the same precursor of Gibberellin (GA). Therefore, in this study, a GA inhibitor, Daminozide (0, 10, 20 ppm) was used to block ent-kaurenoic acid conversion towards GA synthesis in attempt to increase SG content of stevia propagated in Temporary Immersion Bioreactor (TIB). Daminozide in 10 mg/L was observed to be the optimum concentration which increased biomass weight and SG content (stevioside and rebaudioside A) up to 40%. The treatment also increased transcripts accumulation of genes enrolled in SG biosynthesis, such as SrKA13H, SrUGT85C2, and SrUGT76G1, indicating SG pathway become more active due to the inhibition of GA pathway. Furthermore, the inhibition of GA was also indicated by the upregulated expression of GA biosynthesis gene (GA3ox) as the result of feedback regulation, and the downregulated expression of GA catabolism gene (GA2ox2) as the result of feed-forward regulation caused by inhibitor treatment.

Catalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides

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.

Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk

Steviol glycosides are used in food and beverages worldwide as natural sweeteners, serving as a low-calorie sugar substitute. The acceptable daily intake of steviol is 0–4 mg/kg body weight. The rising demand for dairy products has led to a corresponding increase in the use of steviol glycosides in such products. Therefore, it is important to analyze the levels of steviol glycosides in dairy products. Dairy products have high fat contents and unique emulsion characteristics, conferred by a mixture of fat globules, casein micelles, whey proteins, and numerous other small molecules. These characteristics may interfere with the estimation of steviol glycoside levels; therefore, dairy samples require pretreatment. We aimed to develop an objective test for measuring the levels of steviol glycosides through the development of an efficient pretreatment method. In this study, the steviol glycoside content in dairy products was evaluated by using various methods, and an optimal pretreatment method was determined. We used high-performance liquid chromatography to assess the selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, and recovery rate. Calibration curves were linear in the range of 1–50 mg/kg, with a coefficient of determination of ≥0.999. The limit of detection and limit of quantification were in the ranges of 0.11–0.56 and 0.33–1.69 mg/kg, respectively. The relative standard deviation (%) represents the precision of a measurement. The RSD relative standard deviationof recovery varied between 0.16% and 2.83%, and recovery of the analysis varied between 83.57% and 104.84%. These results demonstrate the reliability of the method for measuring the steviol glycoside content. This method can be used for the simple pretreatment of steviol glycosides and can provide an accurate determination of steviol glycoside content in emulsified food matrices, such as dairy products.

The chromosome-level Stevia genome provides insights into steviol glycoside biosynthesis

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.

Molecular Networking Leveraging the Secondary Metabolomes Space of Halophila stipulaceae (Forsk.) Aschers. and Thalassia hemprichii (Ehrenb. ex Solms) Asch. in Tandem with Their Chemosystematics and Antidiabetic Potentials

The Red Sea is one of the most biodiverse aquatic ecosystems. Notably, seagrasses possess a crucial ecological significance. Among them are the two taxa Halophila stipulacea (Forsk.) Aschers., and Thalassia hemprichii (Ehrenb. ex Solms) Asch., which were formally ranked together with the genus Enhalus in three separate families. Nevertheless, they have been recently classified as three subfamilies within Hydrocharitaceae. The interest of this study is to explore their metabolic profiles through ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS/MS) analysis in synergism with molecular networking and to assess their chemosystematics relationship. A total of 144 metabolites were annotated, encompassing phenolic acids, flavonoids, terpenoids, and lipids. Furthermore, three new phenolic acids; methoxy benzoic acid-O-sulphate (16), O-caffeoyl-O-hydroxyl dimethoxy benzoyl tartaric acid (26), dimethoxy benzoic acid-O-sulphate (30), a new flavanone glycoside; hexahydroxy-monomethoxy flavanone-O-glucoside (28), and a new steviol glycoside; rebaudioside-O-acetate (96) were tentatively described. Additionally, the evaluation of the antidiabetic potential of both taxa displayed an inherited higher activity of H. stipulaceae in alleviating the oxidative stress and dyslipidemia associated with diabetes. Hence, the current research significantly suggested Halophila, Thalassia, and Enhalus categorization in three different taxonomic ranks based on their intergeneric and interspecific relationship among them and supported the consideration of seagrasses in natural antidiabetic studies.

Instant Drink Powder Development from Lotus Root with Probiotic Supplement using Foam Mat Drying Method

In this study, we developed instant drink powder of lotus root water using steviol glycoside as a non-nutritive sweetener at different concentrations (0.01, 0.03 and 0.05% w/v) and flavor (Chrysanthemum, Pandan leaves and Roselle) addition on color, pH, %TA, TSS and organoleptic quality properties. The additional of steviol glycoside did not show any effects on physicochemical properties. Results show that 0.03% steviol glycoside and Pandan leaves addition exhibited the highest score of sensorial features significantly. Moreover, this formula was made as foam-mat dried to obtain lotus root supplemented with probiotics powders. The effects of composition ratio of methylcellulose: egg albumin (1:1, 1:2, 1.5:1 and 1.5:2) on the lotus root foam properties were investigated. It was observed that increase in the concentration of methylcellulose decreased foam stability and foam density. Foams were prepared from Lactobacillus plantarum M29 and Peddiococcus pentosaceus MG12 with lotus root water by adding the different concentrations of foaming agents at a whipping time of 15 min. The composition ratio of methylcellulose: egg albumin (1.5:1) provided preferable foam characteristics. The greatest resulting foams were dried at 60 °C for 3 h. The lotus root with probiotics instant drink was able to possess the antioxidant activity and viable probiotics count (more than 7 log CFU/g) during storage at both refrigeration and room temperature for a month.