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.

Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4–4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

Interactive Effects of Arbuscular Mycorrhizal Inoculation with Nano Boron, Zinc, and Molybdenum Fertilization on Stevioside Contents of Stevia (Stevia rebaudiana, L.) Plants

Stevia (Stevia rebaudiana, L.) is receiving increasing global interest as a diabetes-focused herb associated with zero-calorie stevioside sweetener glycoside production. This study was conducted to determine whether the arbuscular mycorrhiza (AM), as a biofertilizer integrated with nano boron (B), zinc (Zn), and molybdenum (Mo), would improve stevia growth and stevioside content. A factorial experiment with four replicates was conducted to evaluate the effect of AM at 0, 150, and 300 spore/g soil and three nano microelements B at 100 mg/L, Zn at 100 mg/L, and Mo at 40 mg/L on growth performance, stevioside, mineral contents, and biochemical contents of stevia. Results indicated that the combination of AM at 150 and B at 100 mg/L significantly increased plant height, number of leaves, fresh and dry-stem, and herbal g/plant during the 2019 and 2020 growing seasons. Chlorophyll content was increased by the combination between AM at 150 spore/g soil and B at 100 mg/L during both seasons. Stevioside content in leaves was increased by AM at 150 spore/g soil and B at 100 mg/L during the second season. In addition, N, P, K, Zn, and B in the leaf were increased by applying the combination of AM and nano microelements. Leaf bio constituent contents were increased with AM at 150 spore/g soil and B at 100 mg/L during both seasons. The application of AM and nano B can be exploited for high growth, mineral, and stevioside contents as a low-calorie sweetener product in stevia.

D-Tagatose Feeding Reduces the Risk of Sugar-Induced Exacerbation of Myocardial I/R Injury When Compared to Its Isomer Fructose

It is known that fructose may contribute to myocardial vulnerability to ischemia/reperfusion (I/R) injury. D-tagatose is a fructose isomer with less caloric value and used as low-calorie sweetener. Here we compared the metabolic impact of fructose or D-tagatose enriched diets on potential exacerbation of myocardial I/R injury. Wistar rats were randomizedly allocated in the experimental groups and fed with one of the following diets: control (CTRL), 30% fructose-enriched (FRU 30%) or 30% D-tagatose-enriched (TAG 30%). After 24 weeks of dietary manipulation, rats underwent myocardial injury caused by 30 min ligature of the left anterior descending (LAD) coronary artery followed by 24 h′ reperfusion. Fructose consumption resulted in body weight increase (49%) as well as altered glucose, insulin and lipid profiles. These effects were associated with increased I/R-induced myocardial damage, oxidative stress (36.5%) and inflammation marker expression. TAG 30%-fed rats showed lower oxidative stress (21%) and inflammation in comparison with FRU-fed rats. Besides, TAG diet significantly reduced plasmatic inflammatory cytokines and GDF8 expression (50%), while increased myocardial endothelial nitric oxide synthase (eNOS) expression (59%). Overall, we demonstrated that D-tagatose represents an interesting sugar alternative when compared to its isomer fructose with reduced deleterious impact not only on the metabolic profile but also on the related heart susceptibility to I/R injury.

IDENTIFIKASI GLIKOSIDA DAN EVALUASI FISIK FORMULA PEMANIS RENDAH KALORI TANAMAN STEVIA (Stevia rebaudiana Bentoni) PADA MINUMAN FUNGSIONAL BIR PLETOK

Instant drinks, which are often consumed by the public, are mostly sweetened drinks that contain high calories and artificialsweeteners and can cause various diseases, such as obesity, cancer, diabetes and others. This can be controlled and prevented by consuminglow-calorie sweeteners. Stevia is a plant that has a sweetness of 300 times that of sucrose, so it can be used as a low-calorie sweetener, suchas in the functional drink Pletok beer. This study aims to identify glycosides and to evaluate the physical evaluation of the low-caloriesweetener formula for Stevia (Stevia rebaudiana B.) in the functional beverage of Bir Pletok. Physical evaluation and hedonism test werecarried out on 20 panelists and 1 expert panelist, then analyzed descriptively. In this study, several tests of instant preparations were carriedout, namely organoleptic test, pH test, specific gravity test, movable volume test and hedonism test on 20 panelists and 1 expert panelist. Theresults of the research that have been done show that the leaves of Stevia (Stevia rebaudiana B.) contain diterpenoid glycosides and based onall test preparations the best formula of the three formulas is Formula II. Keywords: Sweeteners, Stevia, Functional Drinks, Bir Pletok, Glycosides.

THE USE OF LOW CALORIE SWEETENER STEVIA IN VELVA TOMATO (Lycopersicum esculentum mill)

<p><em>The purpose of this research was to determine the effect of velva tomato formula with different proportions of sugar and stevia on the physical, chemical, and sensory of tomato velva. The best tomato velva with sweetener stevia was then determined based on the physical, chemical and sensory characteristics. This research used a completely randomized design (RAL) of one factor which was the propotions of sweetener stevia (K: 50 g sucrose; F1: 39.5 g sucrose and 0.75 g stevia; F2: 29 g sucrose and 1.5g stevia; F3: 19.5 g sucrose and 2.25 g stevia; and F4: 8 g sucrose and 3 g stevia). The result show tat propotions of stevia significantly affects physical characteristics (total dissolved solids, melting power, and overrun), chemical (total calories), and sensory (taste, texture, and overall). While the formula modification has no significant effect on vitamin C and lycopene content. The more proportion of stevia and the less proportion of sucrose reduced total calories, melting power, and total dissolved solids and at the same time increased overrun. The most preferred formula was F1 (39,5g sugar and 0,75g stevia sweetener) which has an overrun of 28.09%, total dissolved solids of 8.43 ^oBrix, melting power of 13.09 minutes and total calories of 328.65 cal/g.</em></p>