Stevia leaves contain glycosides on which biological activity and sweetening capacity has been reported. Besides the main glycosides—stevioside and rebaudioside A—there are minor glycosides that may contribute to th...Stevia leaves contain glycosides on which biological activity and sweetening capacity has been reported. Besides the main glycosides—stevioside and rebaudioside A—there are minor glycosides that may contribute to the activity and thus it is important to quantify them. Rebaudioside D is one of the minor glycoside present in S. rebaudiana leaves and there are no reports of a validated method to quantify it. Therefore a simple and sensitive high performance liquid chromatography (HPLC) method was validated for the determination of rebaudioside D in leaves of Stevia rebaudiana B. grown in the southeast of México. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 ml/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The retention time of rebaudioside D was found to be 3.47 min ± 0.04 (S.D.). The calibration curves were linear over the working range (25 - 150 μg/ml), with correlation coefficient ≥0.99 and determination coefficient ≥0.98. The calculated limit of detection (LOD) was 8.53 μg/ml, while the limit of quantitation (LOQ) was 25.85 μg/ml. The percent recoveries of fortified samples were 100% ± 10% and precision relative standard deviation was ≤2.79%. The criteria of validation showed accuracy, linearity, and precision;therefore the method is suitable for quantitative analysis of rebaudioside D in Stevia rebaudiana leaves. Rebaudioside D content (g/100g) in Morita II and Criolla varieties grown in the southeast of Mexico were 0.43 and 0.46, respectively with no significant differences (p > 0.05) between them.展开更多
The equilibrium solubility of Rebaudioside A(Reb A)FormⅡin binary mixtures of methanol/ethanol and ethyl acetate was quantitatively determined within the temperature range of 283.15—328.15 K at ambient pressure.The ...The equilibrium solubility of Rebaudioside A(Reb A)FormⅡin binary mixtures of methanol/ethanol and ethyl acetate was quantitatively determined within the temperature range of 283.15—328.15 K at ambient pressure.The experimental findings indicate a positive correlation between the solubility of Reb A(FormⅡ)and both the temperature and the methanol/ethanol content in the solvent system.To describe the solubility data,six distinct models were employed:the modified Apelblat equation,theλh model,the combined nearly ideal binary solvent/Redlich—Kister(CNIBS/R—K)model,the van't HoffJouyban-Acree(VJA)model,the Apelblat-Jouyban-Acree(AJA)model,and the non-random two-liquid(NRTL)model.The combined nearly ideal binary solvent/Redlich—Kister model exhibited the most precise fit for solubility in methanol+ethyl acetate mixtures,reflected by an average relative deviation(ARD)of 0.0011 and a root mean square deviation(RMSD)of 12×10^(-7).Conversely,for ethanol+ethyl acetate mixtures,the modified Apelblat equation provided a superior correlation(ARD=0.0014,RMSD=4×10^(-7)).Furthermore,thermodynamic parameters associated with the dissolution of Reb A(FormⅡ),including enthalpy,entropy,and the Gibbs energy change,were inferred from the data.The findings underscore that the dissolution process is predominantly endothermic across the solvent systems examined.Notably,the entropy changes appear to have a significant influence on the Gibbs free energy associated with the dissolution of Reb A(FormⅡ),suggesting that entropic factors may play a pivotal role in the studied systems.展开更多
Stevia rebaudiana is a plant with high sweetening capacity due to its content of glycosides, mainly stevioside and rebaudioside A. Several techniques have been used to determine the concentrations of glycosides in Ste...Stevia rebaudiana is a plant with high sweetening capacity due to its content of glycosides, mainly stevioside and rebaudioside A. Several techniques have been used to determine the concentrations of glycosides in Stevia, although an HPLC method is recommended by the FAO/WHO-JECFA. Varieties of Stevia have been recently grown in Mexico, with no previous report of glycosides by a validated method. The aim of this study was to validate an isocratic HPLC method for content determination of main glycosides in the leaves of Stevia cultivated in Mexico. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium-phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 mL/min. Rebaudioside A and stevioside were determined in two Stevia varieties: Morita II and Criolla, and also validation parameters were calculated. Rebaudioside A content (g/100g) in Morita II was 15.15 ± 0.02 while stevioside was 3.97 ± 0.003;in the case of Criolla they were 4.03 ± 0.01 and 8.80 ± 0.14, respectively (p < 0.001). The recoveries of fortified samples were 100% ± 10% and precision RSD was ≤6.27%. The criteria of validation showed accuracy, linearity (≥0.99), and precision;therefore, the determination of glycosides was performed with reliability.展开更多
Steviol glycosides(SGs)from Stevia rebaudiana Bertoni is a widely used sweetener with the advantage of high sweet and low in calories.Stevioside(ST)and Rebaudioside A(Reb A)with a bitter aftertaste are the main sweete...Steviol glycosides(SGs)from Stevia rebaudiana Bertoni is a widely used sweetener with the advantage of high sweet and low in calories.Stevioside(ST)and Rebaudioside A(Reb A)with a bitter aftertaste are the main sweetening components in SGs.Reb M is a highly potency sweetener with almost no bitter aftertaste but is found in low content in S.rebaudiana Bertoni.In this study,the pre-and post-catalytic differences of stevioside gly-cosyltransferase SrUGT76G1 were investigated through long-time molecular dynamics simulations.SrUGT76G1 was mutated to enhance its efficiency in catalyzing the synthesis of Reb D to Reb M by identifying the enzyme mutation site through an integrated three-in-one approach that combines hinge region analysis,coevolutionary conservation,and energy calculations.The kcat and k_(cat)/K_(m) values of the M88V mutant with Reb D as substrate were 2.18-fold and 4.85-fold higher than those of wild-type SrUGT76G1,respectively.In addition,the M88V mutant coupled with sucrose synthase StSUS1 increased the yield of Reb M synthesis from Reb D to 92.40%.The present study provides an effective method for rapid search for mutation sites in the enzyme and for increasing the yield of Reb M.展开更多
Rebaudioside M8(Reb M8),a high-potency sweetener derived from natural Rebaudioside D found in Stevia rebaudiana,exhibits favorable organoleptic properties as an ideal sugar replacement candidate.However,the industrial...Rebaudioside M8(Reb M8),a high-potency sweetener derived from natural Rebaudioside D found in Stevia rebaudiana,exhibits favorable organoleptic properties as an ideal sugar replacement candidate.However,the industrial biosynthesis of Reb M8 is hindered by the poor thermostability of its key biocatalyst,glycosyltransferase UGT94E13,limiting its application in long-term or high-temperature processes.In this study,a combinatorial strategy integrating computer-aided rational design as well as structural and evolutionary conservation analyses is utilized to enhance the thermostability of a catalytic-efficient UGT94E13-F169G/I185G(M0)to a superior variant M0-T102D/V110I/F208Y/N251E(M5)with a 5.4℃ increase in melting temperature(T_(m)),a 1.08-fold improvement in catalytic activity,and a 14.8-fold extension of half-life relative to M0.The simultaneous improvement of activity and stability is well explained by molecular dynamics simulations and structural analyses,revealing flexibility reduction,hydrophobic packing improvement,salt bridge formation,and electrostatics optimization as key factors for the improved thermal resilience.Furthermore,in a cascade biocatalytic system coupled with sucrose synthase AtSuSy,M5 enabled sustained biosynthesis and highproduction of Reb M8(35.30 g/L)under optimized fed-batch conditions.These findings establish M5 as a robust and scalable biocatalyst that overcomes the inherent activity-stability trade-off of glycosyltransferase UGT94E13,advancing the biomanufacturing potential of promising sweeteners in food and health industries.展开更多
Steviol glycoside(SG),derived from Stevia rebaudiana leaves,is the third-largest global source of sweetener.However,its lingering bitterness and metallic aftertaste limit its applications.Enzymatic glycosylation of na...Steviol glycoside(SG),derived from Stevia rebaudiana leaves,is the third-largest global source of sweetener.However,its lingering bitterness and metallic aftertaste limit its applications.Enzymatic glycosylation of natural SGs is a promising approach for resolving this flavor-related issue.Herein,a novel monoβ-1,6-glycosylated rebaudioside D(Reb D)derivative Reb M8 was selectively produced from Reb D using glycosyltransferase UGT94E13 from Gardenia jasminoides.Next,the sweetness and bioactivity of inhibitory effects of this derivative on the inflammatory factor tumor necrosis factor-alpha(TNF-α),involved in various diseases,were primarily studied.Electronic tongue analysis indicated that the construction of a β-1,6-glucoside bond at the C-13 position of Reb D improved sweetness.Furthermore,Reb M8 exhibited inhibitory effects on TNF-α,implying its potential application in the treatment of various diseases.Overall,this study presents a selective method for producing a new SG Reb M8 with improved sweetness and the ability to inhibit TNF-α,which makes it a promising candidate for several applications in food and pharmaceutical industries.展开更多
The adsorptive capacity and selectivity of a novel adsorbent with pyridyl group toward stevia glycosides were studied. The effect of polarity and physical structure of the sorbent on the selectivity was investigated i...The adsorptive capacity and selectivity of a novel adsorbent with pyridyl group toward stevia glycosides were studied. The effect of polarity and physical structure of the sorbent on the selectivity was investigated in detail. Two separation methods were applied in the enrichment of rebaudioside A(RA). They were selective elution using methanol or ethanol solution as solvent, and dynamic chromatographic separation using pyridyl resin with high selectivity. Results show that the chromatographic separation method can effectively enrich RA from stevia glycoside with high content of stevioside.展开更多
Natural sweetener rebaudioside M(Reb M),with high sweetness,good taste profile and zero-calorie,has abundant market potential.Currently,it is mainly from the glycosylation of rebaudioside D(Reb D)by glycosyltransferas...Natural sweetener rebaudioside M(Reb M),with high sweetness,good taste profile and zero-calorie,has abundant market potential.Currently,it is mainly from the glycosylation of rebaudioside D(Reb D)by glycosyltransferase UGT76G1.However,its poor thermostability hampers the large-scale preparation of Reb M in industry.In this study,through computer-aided target mutagenesis and structure analysis,a four-point mutant M3(I30M/K53A/R141P/G349P)of UGT76G1-T284S/M88L/L200A(M0)with increased melting temperature of 6.89℃ and 1.16-fold improvement of catalytic activity is obtained from the limited mutant library.Based on molecular dynamics simulations and molecular mechanism analysis,it reveals that the improved thermostability is attributed to tighter core packing,redistribution of surface electrostatic charges and higher loop rigidity.Importantly,this variant increases the yield of Reb M to 90.60%from 25 mM Reb D in a fed-batch reaction at 45℃.This work not only provides the thermostable M3 for potential scale-up preparation of Reb M,but also develops robust combinatorial strategies to enhance the enzyme thermostability.展开更多
An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige...An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.展开更多
Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on ...Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.展开更多
Primates have been found to differ widely in their taste perception and studies suggest that a coevolution between plant species bearing a certain taste substance and primate species feeding on these plants may contri...Primates have been found to differ widely in their taste perception and studies suggest that a coevolution between plant species bearing a certain taste substance and primate species feeding on these plants may contribute to such between-species differences. Considering that only platyrrhine primates, but not catarrhine or prosimian primates, share an evolutionary history with the neotrop- ical plant Stevia rebaudiana, we assessed whether members of these three primate taxa differ in their ability to perceive and/or in their sensitivity to its two quantitatively predominant sweet- tasting substances. We found that not only neotropical black-handed spider monkeys, but also paleotropical black-and-white ruffed lemurs and Western chimpanzees are clearly able to perceive stevioside and rebaudioside A. Using a two-bottle preference test of short duration, we found that Ateles geoffroyi preferred concentrations as low as 0.05 mM stevioside and 0.01 mM rebaudioside A over tap water. Taste preference thresholds of Pan troglodytes were similar to those of the spider monkeys, with 0.05 mM for stevioside and 0.03 mM for rebaudioside A, whereas Varecia variegata was slightly less sensitive with a threshold value of 0.1 mM for both substances. Thus, all three primate species are, similar to human subjects, clearly more sensitive to both steviol glycosides compared to sucrose. Only the spider monkeys displayed concentration-response curves with both stevioside and rebaudioside A which can best be described as an inverted U-shaped function sug- gesting that Ateles geoffroyi, similar to human subjects, may perceive a bitter side taste at higher concentrations of these substances. Taken together, the results of the present study do not support the notion that a co-evolution between plant and primate species may account for between-species differences in taste perception of steviol glycosides.展开更多
A series of porous resins (PYR) with different content of pyridyl group were prepared,and their physical structure and adsorption properties on the stevia glycosides werestudied in detail. Afler the selective adsorpti...A series of porous resins (PYR) with different content of pyridyl group were prepared,and their physical structure and adsorption properties on the stevia glycosides werestudied in detail. Afler the selective adsorption of PYR resins, the rebaudioside A couldbe separated hem the other components of the stevia glycosides, and a new methodforobtaining pure rebaudioside A is reported.展开更多
The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia ...The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia is a polyannual crop (5 - 7 years) with a vegetative period lasting from April-May until October, during which time the crop can be harvested two or three times. This research focuses on the need for knowledge about Stevia’s response to temperate climates in Southern Europe. Two field assays were established from June to October 2013 at two sites in Navarra (Spain). The biomass and content of the two major SG, stevioside (ST) and rebaudioside A (RA), were measured using High Performance Liquid Chromatography (HPLC) in 66 cloned plants, at different developmental stages. Although the results from the two sites showed different SG leaf content dynamics during the plant growth, the optimum harvest date at both sites coincided with the bud-flowering stage at the beginning of September (around 96 days after planting), when a ST yield of 27 g·m-2 was reached. These results show Stevia’s potential as a commercial crop for stevioside production in northern Spain.展开更多
Some hydrophobic (including both the non\|polar and polar) and hydrophilic polymer adsorbents were designed and synthesized, and their adsorption properties and adsorption mechanism toward stevia glycosides were studi...Some hydrophobic (including both the non\|polar and polar) and hydrophilic polymer adsorbents were designed and synthesized, and their adsorption properties and adsorption mechanism toward stevia glycosides were studied in great detail. The skeleton structure and polarity of the resins had effect on the adsorption capacity and the selectivity properties during the adsorption of stevioside and rebaudioside A. A sweetener with high rebaudioside A content was isolated by using the adsorption selectivity of the polar resins.展开更多
基金This work was supported by Fondos Fiscales-INIFAP through projects named“Desarrollo de productos alimenticios elaborados con hoja de Stevia rebaudiana Bertoni”Also,I.A.G.received a CONACYT’s scholarship during postgraduate studies.
文摘Stevia leaves contain glycosides on which biological activity and sweetening capacity has been reported. Besides the main glycosides—stevioside and rebaudioside A—there are minor glycosides that may contribute to the activity and thus it is important to quantify them. Rebaudioside D is one of the minor glycoside present in S. rebaudiana leaves and there are no reports of a validated method to quantify it. Therefore a simple and sensitive high performance liquid chromatography (HPLC) method was validated for the determination of rebaudioside D in leaves of Stevia rebaudiana B. grown in the southeast of México. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 ml/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The retention time of rebaudioside D was found to be 3.47 min ± 0.04 (S.D.). The calibration curves were linear over the working range (25 - 150 μg/ml), with correlation coefficient ≥0.99 and determination coefficient ≥0.98. The calculated limit of detection (LOD) was 8.53 μg/ml, while the limit of quantitation (LOQ) was 25.85 μg/ml. The percent recoveries of fortified samples were 100% ± 10% and precision relative standard deviation was ≤2.79%. The criteria of validation showed accuracy, linearity, and precision;therefore the method is suitable for quantitative analysis of rebaudioside D in Stevia rebaudiana leaves. Rebaudioside D content (g/100g) in Morita II and Criolla varieties grown in the southeast of Mexico were 0.43 and 0.46, respectively with no significant differences (p > 0.05) between them.
基金supported by the National Key Research and Development Program of China(2021YFC2103800)the National Natural Science Foundation of China(U21A20301)the Research Funds of Institute of Zhejiang University-Quzhou(IZQ2022RCZX004 and IZQ2021RCZX015)。
文摘The equilibrium solubility of Rebaudioside A(Reb A)FormⅡin binary mixtures of methanol/ethanol and ethyl acetate was quantitatively determined within the temperature range of 283.15—328.15 K at ambient pressure.The experimental findings indicate a positive correlation between the solubility of Reb A(FormⅡ)and both the temperature and the methanol/ethanol content in the solvent system.To describe the solubility data,six distinct models were employed:the modified Apelblat equation,theλh model,the combined nearly ideal binary solvent/Redlich—Kister(CNIBS/R—K)model,the van't HoffJouyban-Acree(VJA)model,the Apelblat-Jouyban-Acree(AJA)model,and the non-random two-liquid(NRTL)model.The combined nearly ideal binary solvent/Redlich—Kister model exhibited the most precise fit for solubility in methanol+ethyl acetate mixtures,reflected by an average relative deviation(ARD)of 0.0011 and a root mean square deviation(RMSD)of 12×10^(-7).Conversely,for ethanol+ethyl acetate mixtures,the modified Apelblat equation provided a superior correlation(ARD=0.0014,RMSD=4×10^(-7)).Furthermore,thermodynamic parameters associated with the dissolution of Reb A(FormⅡ),including enthalpy,entropy,and the Gibbs energy change,were inferred from the data.The findings underscore that the dissolution process is predominantly endothermic across the solvent systems examined.Notably,the entropy changes appear to have a significant influence on the Gibbs free energy associated with the dissolution of Reb A(FormⅡ),suggesting that entropic factors may play a pivotal role in the studied systems.
基金supported by Programa de Mejoramiento al Profesorado-PROMEP-SEPFondos Fiscales-INIFAP
文摘Stevia rebaudiana is a plant with high sweetening capacity due to its content of glycosides, mainly stevioside and rebaudioside A. Several techniques have been used to determine the concentrations of glycosides in Stevia, although an HPLC method is recommended by the FAO/WHO-JECFA. Varieties of Stevia have been recently grown in Mexico, with no previous report of glycosides by a validated method. The aim of this study was to validate an isocratic HPLC method for content determination of main glycosides in the leaves of Stevia cultivated in Mexico. HPLC method was performed using a C18 column (250 mm × 4.6 mm, 5 μm) and UV detector set at 210 nm. The mobile phase consisted of 32:68 (v/v) mixture of acetonitrile and sodium-phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 mL/min. Rebaudioside A and stevioside were determined in two Stevia varieties: Morita II and Criolla, and also validation parameters were calculated. Rebaudioside A content (g/100g) in Morita II was 15.15 ± 0.02 while stevioside was 3.97 ± 0.003;in the case of Criolla they were 4.03 ± 0.01 and 8.80 ± 0.14, respectively (p < 0.001). The recoveries of fortified samples were 100% ± 10% and precision RSD was ≤6.27%. The criteria of validation showed accuracy, linearity (≥0.99), and precision;therefore, the determination of glycosides was performed with reliability.
基金supported by the fund of Beijing Natural Science Foundation(No.6242001)the fund of the Cultivation Project of the Double First-Class Disciplines of Food Science and Engineering,Beijing Technology and Business University(No.BTBUYXTD202207)+1 种基金Discipline Construction——Food Science and Engineering(No.SPKX-202204)the Research Foundation for Youth Scholars of Beijing Technology and Business University(No.QNJJ2022-06)‘Xinjiang’Two Zones'Science and Technology Development Plan Project(No.2022LQ02003).
文摘Steviol glycosides(SGs)from Stevia rebaudiana Bertoni is a widely used sweetener with the advantage of high sweet and low in calories.Stevioside(ST)and Rebaudioside A(Reb A)with a bitter aftertaste are the main sweetening components in SGs.Reb M is a highly potency sweetener with almost no bitter aftertaste but is found in low content in S.rebaudiana Bertoni.In this study,the pre-and post-catalytic differences of stevioside gly-cosyltransferase SrUGT76G1 were investigated through long-time molecular dynamics simulations.SrUGT76G1 was mutated to enhance its efficiency in catalyzing the synthesis of Reb D to Reb M by identifying the enzyme mutation site through an integrated three-in-one approach that combines hinge region analysis,coevolutionary conservation,and energy calculations.The kcat and k_(cat)/K_(m) values of the M88V mutant with Reb D as substrate were 2.18-fold and 4.85-fold higher than those of wild-type SrUGT76G1,respectively.In addition,the M88V mutant coupled with sucrose synthase StSUS1 increased the yield of Reb M synthesis from Reb D to 92.40%.The present study provides an effective method for rapid search for mutation sites in the enzyme and for increasing the yield of Reb M.
基金supported by the Natural Science Foundation of Jiangsu Province(BK20202002)National Natural Science Foundation of China(22207044,22477047,32270082)the Fundamental Research Funds for the Central Universities(JUSRP202404012,JUSRP124020).
文摘Rebaudioside M8(Reb M8),a high-potency sweetener derived from natural Rebaudioside D found in Stevia rebaudiana,exhibits favorable organoleptic properties as an ideal sugar replacement candidate.However,the industrial biosynthesis of Reb M8 is hindered by the poor thermostability of its key biocatalyst,glycosyltransferase UGT94E13,limiting its application in long-term or high-temperature processes.In this study,a combinatorial strategy integrating computer-aided rational design as well as structural and evolutionary conservation analyses is utilized to enhance the thermostability of a catalytic-efficient UGT94E13-F169G/I185G(M0)to a superior variant M0-T102D/V110I/F208Y/N251E(M5)with a 5.4℃ increase in melting temperature(T_(m)),a 1.08-fold improvement in catalytic activity,and a 14.8-fold extension of half-life relative to M0.The simultaneous improvement of activity and stability is well explained by molecular dynamics simulations and structural analyses,revealing flexibility reduction,hydrophobic packing improvement,salt bridge formation,and electrostatics optimization as key factors for the improved thermal resilience.Furthermore,in a cascade biocatalytic system coupled with sucrose synthase AtSuSy,M5 enabled sustained biosynthesis and highproduction of Reb M8(35.30 g/L)under optimized fed-batch conditions.These findings establish M5 as a robust and scalable biocatalyst that overcomes the inherent activity-stability trade-off of glycosyltransferase UGT94E13,advancing the biomanufacturing potential of promising sweeteners in food and health industries.
基金supported by the National Key R&D Program of China(2018YFA0901700)the Natural Science Foundation of Jiangsu Province(BK20202002).
文摘Steviol glycoside(SG),derived from Stevia rebaudiana leaves,is the third-largest global source of sweetener.However,its lingering bitterness and metallic aftertaste limit its applications.Enzymatic glycosylation of natural SGs is a promising approach for resolving this flavor-related issue.Herein,a novel monoβ-1,6-glycosylated rebaudioside D(Reb D)derivative Reb M8 was selectively produced from Reb D using glycosyltransferase UGT94E13 from Gardenia jasminoides.Next,the sweetness and bioactivity of inhibitory effects of this derivative on the inflammatory factor tumor necrosis factor-alpha(TNF-α),involved in various diseases,were primarily studied.Electronic tongue analysis indicated that the construction of a β-1,6-glucoside bond at the C-13 position of Reb D improved sweetness.Furthermore,Reb M8 exhibited inhibitory effects on TNF-α,implying its potential application in the treatment of various diseases.Overall,this study presents a selective method for producing a new SG Reb M8 with improved sweetness and the ability to inhibit TNF-α,which makes it a promising candidate for several applications in food and pharmaceutical industries.
基金Project supported by the National Post-doctoral Foundation of China (97/No.6)the State Key Laboratory of Functional Polymeric Materials for Adsorption and Separation (SKLFPMAS) KF97036, Nankai University
文摘The adsorptive capacity and selectivity of a novel adsorbent with pyridyl group toward stevia glycosides were studied. The effect of polarity and physical structure of the sorbent on the selectivity was investigated in detail. Two separation methods were applied in the enrichment of rebaudioside A(RA). They were selective elution using methanol or ethanol solution as solvent, and dynamic chromatographic separation using pyridyl resin with high selectivity. Results show that the chromatographic separation method can effectively enrich RA from stevia glycoside with high content of stevioside.
基金funded by National Key R&D Program of China(2018YFA0901700)the Natural Science Foundation of Jiangsu Province(BK20202002)National Natural Science Foundation of China(22207044).
文摘Natural sweetener rebaudioside M(Reb M),with high sweetness,good taste profile and zero-calorie,has abundant market potential.Currently,it is mainly from the glycosylation of rebaudioside D(Reb D)by glycosyltransferase UGT76G1.However,its poor thermostability hampers the large-scale preparation of Reb M in industry.In this study,through computer-aided target mutagenesis and structure analysis,a four-point mutant M3(I30M/K53A/R141P/G349P)of UGT76G1-T284S/M88L/L200A(M0)with increased melting temperature of 6.89℃ and 1.16-fold improvement of catalytic activity is obtained from the limited mutant library.Based on molecular dynamics simulations and molecular mechanism analysis,it reveals that the improved thermostability is attributed to tighter core packing,redistribution of surface electrostatic charges and higher loop rigidity.Importantly,this variant increases the yield of Reb M to 90.60%from 25 mM Reb D in a fed-batch reaction at 45℃.This work not only provides the thermostable M3 for potential scale-up preparation of Reb M,but also develops robust combinatorial strategies to enhance the enzyme thermostability.
文摘An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.
文摘Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.
文摘Primates have been found to differ widely in their taste perception and studies suggest that a coevolution between plant species bearing a certain taste substance and primate species feeding on these plants may contribute to such between-species differences. Considering that only platyrrhine primates, but not catarrhine or prosimian primates, share an evolutionary history with the neotrop- ical plant Stevia rebaudiana, we assessed whether members of these three primate taxa differ in their ability to perceive and/or in their sensitivity to its two quantitatively predominant sweet- tasting substances. We found that not only neotropical black-handed spider monkeys, but also paleotropical black-and-white ruffed lemurs and Western chimpanzees are clearly able to perceive stevioside and rebaudioside A. Using a two-bottle preference test of short duration, we found that Ateles geoffroyi preferred concentrations as low as 0.05 mM stevioside and 0.01 mM rebaudioside A over tap water. Taste preference thresholds of Pan troglodytes were similar to those of the spider monkeys, with 0.05 mM for stevioside and 0.03 mM for rebaudioside A, whereas Varecia variegata was slightly less sensitive with a threshold value of 0.1 mM for both substances. Thus, all three primate species are, similar to human subjects, clearly more sensitive to both steviol glycosides compared to sucrose. Only the spider monkeys displayed concentration-response curves with both stevioside and rebaudioside A which can best be described as an inverted U-shaped function sug- gesting that Ateles geoffroyi, similar to human subjects, may perceive a bitter side taste at higher concentrations of these substances. Taken together, the results of the present study do not support the notion that a co-evolution between plant and primate species may account for between-species differences in taste perception of steviol glycosides.
文摘A series of porous resins (PYR) with different content of pyridyl group were prepared,and their physical structure and adsorption properties on the stevia glycosides werestudied in detail. Afler the selective adsorption of PYR resins, the rebaudioside A couldbe separated hem the other components of the stevia glycosides, and a new methodforobtaining pure rebaudioside A is reported.
基金funded through Department of Rural Development of the Navarra Government(project IIM14153.RI1).
文摘The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia is a polyannual crop (5 - 7 years) with a vegetative period lasting from April-May until October, during which time the crop can be harvested two or three times. This research focuses on the need for knowledge about Stevia’s response to temperate climates in Southern Europe. Two field assays were established from June to October 2013 at two sites in Navarra (Spain). The biomass and content of the two major SG, stevioside (ST) and rebaudioside A (RA), were measured using High Performance Liquid Chromatography (HPLC) in 66 cloned plants, at different developmental stages. Although the results from the two sites showed different SG leaf content dynamics during the plant growth, the optimum harvest date at both sites coincided with the bud-flowering stage at the beginning of September (around 96 days after planting), when a ST yield of 27 g·m-2 was reached. These results show Stevia’s potential as a commercial crop for stevioside production in northern Spain.
文摘Some hydrophobic (including both the non\|polar and polar) and hydrophilic polymer adsorbents were designed and synthesized, and their adsorption properties and adsorption mechanism toward stevia glycosides were studied in great detail. The skeleton structure and polarity of the resins had effect on the adsorption capacity and the selectivity properties during the adsorption of stevioside and rebaudioside A. A sweetener with high rebaudioside A content was isolated by using the adsorption selectivity of the polar resins.
