rubusoside and stevioside

The present study aimed to specifically transform stevioside (ST) into rubusoside (RS) through bioconversion with high efficiency, seeking to endow steviol glycosides (SGs) with subtle flavours for commercial acceptability.. An endophytic bacterium named Lelliottia LST-1 was screened and confirmed to specifically convert ST into RS, reaching a conversion rate of 75.4% after response surface optimization. Phylogenetic analysis combined with complete genome sequencing demonstrated that LST-1 was also presumed to be a new species. To further explore the principle and process of biological transformation, the potential beta-glucosidases GH3-1, GH3-2, GH3-3 and GH3-4 were expressed, purified and reacted with SGs. High-performance liquid chromatography revealed that all enzymes hydrolysed ST and generated RS, but substrate specificity analysis indicated that GH3-2 had the highest substrate specificity towards STs and the highest enzyme activity.. The potential β-glucosidase GH3-2 in Lelliottia sp. LST-1 was found to specifically and efficiently convert ST to RS.. The efficient biotransformation of ST into RS will be beneficial to its large-scale production and extensive application in the food and pharmaceutical industries.

Topics: Diterpenes, Kaurane; Glucosides; Phylogeny

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5-6.5 and 30-40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher β-glucosidase activity toward the β-linked glucosidic bond of stevioside than toward other β-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The β-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this β-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product.

Topics: beta-Glucosidase; Diterpenes, Kaurane; Glucosides; Lactic Acid; Lactobacillus plantarum

High content of steviol glycosides in stevia leaves is a cause of their high popularity as. a natural sweetener of various sugar-free food products. Stevioside (13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester) is one of the main steviol glycosides in stevia leaves known for its hydrolytic instability responsible for the formation of simple steviol glucosides (steviolbioside, rubusoside, steviol monoside) and steviol. However, the formation of hydroxy and alkoxy adducts of stevioside and of its hydrolysis products has not yet been reported. The performed experiments prove that water and alkoxy adducts are formed not only during temperature processing of stevioside but also of stevia and stevia-containing food products. Their quantities depend on environment pH, water concentration and food composition. Although they are formed in small amounts their biological activity is unknown and should be recognized.

Topics: Diterpenes, Kaurane; Food Analysis; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Methanol; Plant Leaves; Stevia; Sweetening Agents; Temperature; Water

Enzyme specificity and particularity is needed not only in enzymatic separation methods, but also in enzymatic determination methods for plant compound extraction. Stevioside, rubusoside, and rebaudioside A are natural sweet compounds from plants. These compounds have the same skeleton and only contain different side-chain glucosyl groups, making them difficult to separate. However, enzymes that target diterpenoid compounds and show specific activity for side-chain glucosyl groups are rare. Herein, we report the identification and characterization of an enzyme that can target both diterpenoid compounds and sophorose, namely, β-glucosidase SPBGL1 from Sphingomonas elodea ATCC 31461. SPBGL1 displayed high specificity toward sophorose, and activity toward stevioside, but not rebaudioside A. The stevioside conversion rate was 98%. SPBGL1 also operated at high substrate concentrations, such as in 50% crude steviol glycoside extract. Glucose liberated from stevioside was easy to quantify using the glucose oxidase method, allowing the stevioside content to be determined.

Topics: beta-Glucosidase; Diterpenes, Kaurane; Glucosides; Hydrolysis; Plant Extracts; Recombinant Proteins; Sphingomonas; Substrate Specificity

In order to produce rubusoside, enzymes with preferential specificity for the saccharide sophorose were tested for ability to produce rubusoside from stevioside. We identified BGL1, a β-glucosidase from Streptomyces sp. GXT6, as an enzyme for rubusoside production. Out of several saccharide substrates, BGL1 showed the most affinity to sophorose. This enzyme only hydrolyzes the glucose moiety of the sophoroside at C-13 in stevioside. Production of rubusoside was determined by (1)H and (13)C nuclear magnetic resonance (NMR). Thus, rubusoside was produced from stevioside and the stevioside conversion rate was 98.2 %. The production yield of rubusoside was 78.8 % in 6 h.

Topics: beta-Glucosidase; Diterpenes, Kaurane; Glucans; Glucosides; Magnetic Resonance Spectroscopy; Streptomyces

Two new diterpene glycosides in addition to five known glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 (rebaudioside KA) was shown to be 13-[(O-β-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 2-O-β-d-glucopyranosyl-β-d-glucopyranosyl ester and compound 2, 12-α-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester. Five additional known compounds were identified, rebaudioside E, rebaudioside M, rebaudioside N, rebaudioside O, and stevioside, respectively. Enzymatic hydrolysis of stevioside afforded the known ent-kaurane aglycone 13-hydroxy-ent-kaur-16-en-19-oic acid (steviol) (3). The isolated metabolite 1 possesses the ent-kaurane aglycone steviol (3), while compound 2 represents the first example of the isomeric diterpene 12-α-hydroxy-ent-kaur-16-en-19-oic acid existing as a glycoside in S. rebaudiana. The structures of the isolated metabolites 1 and 2 were determined based on comprehensive 1D- and 2D-NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 has formed, which allowed the acquisition of X-ray diffraction data that confirmed its structure. The structural similarities between the new metabolites and the commercially available stevioside sweeteners suggest the newly isolated metabolites should be examined for their organoleptic properties. Accordingly rebaudiosides E, M, N, O, and KA have been isolated in greater than gram quantities.

Topics: Diterpenes, Kaurane; Glucosides; Minnesota; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Plant Leaves; Stevia; Sweetening Agents

Solubility is an important factor for achieving the desired plasma level of drug for pharmacological response. About 40% of drugs are not soluble in water in practice and therefore are slowly absorbed, which results in insufficient and uneven bioavailability and GI toxicity. Rubusoside (Ru) is a sweetener component in herbal tea and was discovered to enhance the solubility of a number of pharmaceutically and medicinally important compounds, including anticancer compounds. In this study, thirty-one hydrolyzing enzymes were screened for the conversion of stevioside (Ste) to Ru. Recombinant lactase from Thermus thermophiles which was expressed in Escherichia coli converted stevioside to rubusoside as a main product. Immobilized lactase was prepared and used for the production of rubusoside; twelve reaction cycles were repeated with 95.4% of Ste hydrolysis and 49 g L(-1) of Ru was produced. The optimum rubusoside synthesis yield was 86% at 200 g L(-1), 1200 U lactase. The purified 10% rubusoside solution showed increased water solubility of liquiritin from 0.98 mg mL(-1) to 4.70±0.12 mg mL(-1) and 0 mg mL(-1) to 3.42±0.11 mg mL(-1) in the case of teniposide.

Topics: Antineoplastic Agents; Bacterial Proteins; Biological Availability; Diterpenes, Kaurane; Enzyme Stability; Enzymes, Immobilized; Flavanones; Glucosides; Humans; Industrial Microbiology; Lactase; Recombinant Proteins; Solubility; Sweetening Agents; Temperature; Teniposide; Thermus thermophilus

Meeting the rising consumer demand for natural food ingredients, steviol glycosides, the sweet principle of Stevia rebaudiana Bertoni (Bertoni), have recently been approved as food additives in the European Union. As regulatory constraints require sensitive methods to analyze the sweet-tasting steviol glycosides in foods and beverages, a HILIC-MS/MS method was developed enabling the accurate and reliable quantitation of the major steviol glycosides stevioside, rebaudiosides A-F, steviolbioside, rubusoside, and dulcoside A by using the corresponding deuterated 16,17-dihydrosteviol glycosides as suitable internal standards. This quantitation not only enables the analysis of the individual steviol glycosides in foods and beverages but also can support the optimization of breeding and postharvest downstream processing of Stevia plants to produce preferentially sweet and least bitter tasting Stevia extracts.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Food Analysis; Glucosides; Glycosides; Plant Leaves; Reference Standards; Sensitivity and Specificity; Stevia; Sweetening Agents; Tandem Mass Spectrometry

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific β-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other β-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 μL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.

Topics: Aspergillus; Cellulases; Diterpenes, Kaurane; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Substrate Specificity

Catalytic hydrogenation of the three ent-kaurane diterpene glycosides isolated from Stevia rebaudiana, namely rubusoside, stevioside, and rebaudioside-A has been carried out using Pd(OH)₂ and their corresponding dihydro derivatives have been isolated as the products. Synthesis of reduced steviol glycosides was performed using straightforward chemistry and their structures were characterized on the basis of 1D and 2D NMR spectral data and chemical studies. Also, we report herewith the sensory evaluation of all the reduced compounds against their corresponding original steviol glycosides and sucrose for the sweetness property of these molecules.

Topics: Catalysis; Diterpenes, Kaurane; Fungal Proteins; Glucosides; Glycosides; Humans; Hydrogenation; Hydrolysis; Hydroxides; Oxidation-Reduction; Palladium; Polygalacturonase; Sweetening Agents; Taste

The purpose of our study was to separate and identify a bacillus that could convert stevioside specifically. Then we identified the conversion product and studied the conversion capability of the bacillus. We also studied the enzyme with conversion capability and the conversion characteristic of the enzyme.. The bacillus was identified on the basis of morphology features and 16S rDNA sequence analysis. Phylogenetic tree was constructed to determine its taxonomic status. The product was detected and identified by high performance liquid chromatography and liquid chromatography-mass spectrometry methods. We use bacteria media directly to studied the conversion capability of the bacillus, and use resting cells, extracellular fluid and intracellular fluid to convert stevioside to determine the enzyme and made further study to learn its conversion characteristic.. The 16S rDNA sequence of the strain had 99% similarity with Chryseobacterium sp., which was ultimately identified as Chryseobacterium sp., JH. The product of biotransformation was rubusoside and the enzyme that converts stevioside into rubusoside was intracellular enzyme. The conversion rate could reach 100%, obtained 5.7 g/L rubusodide solution after 48 h by bacteria media when the concentration of stevia glycosides was 10 g/L, including 7.2 g/L stevioside.. The isolated strain JH was identified as Chryseobacterium sp. It was a novel strain with high, specific ability to convert stevioside into rubusoside which had potential applications.

Topics: Biotransformation; Chryseobacterium; Diterpenes, Kaurane; DNA, Bacterial; DNA, Ribosomal; Glucosides; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S; Soil Microbiology

Three novel diterpene glycosides were isolated for the first time from the commercial extract of the leaves of Stevia rebaudiana, along with several known steviol glycosides, namely stevioside, rebaudiosides A-F, rubusoside and dulcoside A. The new compounds were identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-15-en-19-oic acid, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-16β-hydroxy-ent-kauran-19-oic acid and 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester on the basis of extensive 2D NMR and MS spectroscopic data as well as chemical studies.

Topics: Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Magnetic Resonance Spectroscopy; Molecular Structure; Plant Extracts; Plant Leaves; Stevia

From the commercial extract of the leaves of Stevia rebaudiana, two new minor diterpene glycosides having α-glucosyl linkage were isolated besides the known steviol glycosides including stevioside, steviolbioside, rebaudiosides A-F, rubusoside and dulcoside A. The structures of the two compounds were identified as 13-[(2-O-(3-α-O-d-glucopyranosyl)-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.

Topics: Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Magnetic Resonance Spectroscopy; Molecular Structure; Stevia

In a search for potential cancer chemopreventive agents from natural resources, stevioside (1), a sweetener, and six related compounds, including two aglycones steviol (6) and isosteviol (7), were screened in an in vitro assay for inhibitory effects on Epstein-Barr virus early antigen activation. Compounds 1, 6 and 7 showed significant activity in this assay and also exhibited strong inhibitory effects in a two-stage carcinogenesis test using mouse skin induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The inhibitory effects of these three compounds were greater than that of glycyrrhizin. Furthermore, these three compounds significantly inhibited mouse skin carcinogenesis initiated by peroxynitrite and promoted by TPA. Their activities were comparable to that of curcumin. These results suggested that 1, as well as 6 and 7, could be valuable as chemopreventive agents for chemical carcinogenesis.

Topics: Animals; Antineoplastic Agents; Carcinogenicity Tests; Chemoprevention; Curcumin; Diterpenes, Kaurane; Glucosides; Glycyrrhizic Acid; Herpesvirus 4, Human; Mice; Skin Neoplasms

1,4-alpha-Glucosylation at the 13-O-glycosyl moiety of stevioside (S) and rubusoside (RU) results in a significant increase of sweetness. Saponification of the 19-COO-beta-glucosyl linkage of S and RU yielded steviolbioside (SB) (= 13-O-beta-sophorosyl-steviol) and steviolmonoside (SM) (= 13-O-beta-glucosyl-steviol), respectively, both of which are poorly soluble in an acetate buffer. It was found that the solubilities of SM and SB in the buffer solution were remarkably increased in the presence of gamma-cyclodextrin (gamma-CD). SB was solubilized in the buffer solution with the aid of gamma-CD, and the solution was subjected to 1,4-alpha-transglucosylation by using a cyclodextrin glucanotransferase-starch system to give a mixture of products which were glucosylated at the 13-O-glycosyl moiety. This mixture was acetylated, and the acetate was subjected to chemical beta-glucosylation of 19-COOH followed by deacetylation to afford compounds which have superior sweetness to S. In the same way, derivatives with superior sweetness were selectively prepared from RU through SM.

Topics: Cyclodextrins; Diterpenes; Diterpenes, Kaurane; gamma-Cyclodextrins; Glucosides; Glycosides; Solubility; Sweetening Agents; Terpenes

Steviol (ent-13-hydroxykaur-16-en-19-oic acid), the aglycone of various plant-derived glycoside sweeteners consumed by human populations, is known to be mutagenic toward Salmonella tymphimurium strain TM677 when metabolically activated using a 9000 x g supernatant fraction derived from the liver of Aroclor 1254-pretreated rats. Mass spectral analysis of this diterpenoid and some analogs revealed characteristic patterns reflecting differential stereochemistry at the C/D rings and variations in the nature of the substituents present. Such information has been used to help identify several in vitro metabolites of steviol in conditions known to produce a mutagenic response, when analyzed by gas chromatography/mass spectrometry. The major pathways of such steviol mammalian metabolism proved to be allylic oxidation and epoxidation. 15-Oxosteviol, a product of oxidation of the major steviol metabolite, 15alpha-hydroxysteviol, was found to be a direct-acting mutagen [corrected].

Topics: Biotransformation; Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Mass Spectrometry; Mutagens; Sweetening Agents; Terpenes