Potassium deficiency inhibits steviol glycosides synthesis by limiting leaf sugar metabolism in stevia(Stevia rebaudiana Bertoni)plants

The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K)fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.

Improvement of the Assay Method for Steviol Glycosides in the JECFA Specifications

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.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Synthesis of an IS and Steviol Glycoside Analysis by a Validated Internal Standard Method

The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of steviol glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.

甜菊糖苷的生物学功能及其在畜禽生产中的应用研究进展

甜菊糖苷是一类提取于甜叶菊的四环二萜类糖苷物质,被认为是一种天然、安全、低热量、高甜度的甜味剂。因其具有良好的抗氧化、抗炎、免疫调节及促生长的作用,可作为新型饲料添加剂应用于畜禽生产。本文主要综述了甜菊糖苷的化学结构、提取方法以及味觉评价、甜菊糖苷在机体的水解代谢过程、甜菊糖苷的生物学功能及其在畜禽生产中的相关应用进展,以期为甜菊糖苷在畜禽生产中的合理开发和应用提供参考。

青贮甜菊叶内甜菊糖苷提取行为研究

【目的】为促进甜菊叶收储方式革新,打造低成本甜菊糖原料叶生产体系。【方法】本研究对不同青贮时长甜菊叶内甜菊糖苷提取行为进行分析。【结果】当青贮时间为3、7、15、21、30、60 d,甜菊叶所含各甜菊糖苷的常温水浸泡压榨提取效率随青贮时间延长逐渐增高,提取后各甜菊糖苷残余量随青贮时间延长而下降;70℃水浴浸泡提取4次后,日晒干叶中甜菊苷(Stevioside, STV)和瑞鲍迪苷(Rebaudiosides)系列(RA、RF、RC和RB等)甜菊糖苷残余量为2.7%~6.3%,青贮6月后直接浸提的各甜菊糖苷残余量为8.2%~13.3%,青贮6月烘干后再浸提的各甜菊糖苷残余量为12.2%~30.8%。【结论】本文除对影响青贮甜菊叶内各甜菊糖苷提取效率因素进行了探讨外,还基于研究结果对甜菊湿法收贮必要性和可行性进行了分析,为建立甜菊低成本收储方式提供参考。

甜叶菊主要功能性成分研究进展

甜叶菊是一种重要的中草药、新型糖料作物和具有极高价值的经济作物。其富含糖苷类化合物、黄酮类化合物、绿原酸类化合物和其他多种功能性成分,具有抗氧化、抗菌、抗癌症、降血压、降血脂、降血糖、防龋齿等多种药理活性,日益受到人们的关注,目前在全球范围内广泛应用于食品、药品、保健品和化妆品等多个领域,具有极高的经济价值和广阔的市场前景。本文对甜叶菊主要功能性成分糖苷类化合物、黄酮类化合物和绿原酸类化合物的组成、检测、提取、分离纯化、功能活性、开发利用等方面进行了综述,并对其目前存在的问题和发展前景进行了展望,以期为我国甜叶菊资源的进一步开发、研究和利用提供一定的参考和借鉴。

Isolation and functional analysis of SrMYB1,a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.

甜菊醇糖苷生物合成关键基因的导入和鉴定分析

甜叶菊(Stevia rebaudiana Bertoni)生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点,被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1,构建植物基因过量表达载体,以单独或组合的形式将这些基因导入到甜叶菊中,获得转基因植株。与野生型对照植株相比,单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高,总糖苷、莱包迪苷A含量及占比没有明显变化;单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低,而甜菊醇双糖苷含量显著增加;单独导入SrUGT76G1的转基因植株中,总糖苷含量显著提高,莱包迪苷A含量达到10%以上,比对照提高了2倍,而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似,其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达,培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。

施钾对甜叶菊糖苷跃变期生理指标和产量品质的影响

为提高河西绿洲灌区甜叶菊产量和品质,提升该区甜叶菊市场竞争力。以谱星6号为试验材料,比较了不同施钾水平(K2O 0、60、90、120、150 kg/hm^(2))对甜叶菊糖苷跃变期生理指标以及产量品质的影响。结果表明,不同施钾处理下,甜叶菊整个糖苷跃变期叶片各生理指标含量达到峰值的时间有所不同,叶片SOD活力、含钾量均在现蕾前5 d达到最高,可溶性蛋白、SPAD值、含氮磷量、总苷含量以及叶片干物质量在现蕾初期达到最高,POD活力、可溶性糖含量在现蕾后5 d达到最高,以上各指标均以施用K2O 120 kg/hm^(2)处理表现最佳。其中总苷含量最高,达128.4 g/kg,较不施钾肥增加14.19%;产量也最高,达7007.64 kg/hm^(2),较不施钾肥增加57.24%;净收益达5.26万元/hm^(2),比不施钾多3.40万元/hm^(2)。通过甜叶菊叶片理化指标与产量品质的相关性分析,以上各生理指标与产量品质均呈现显著或者极显著的正相关关系。在当地甜叶菊生产中施用K2O 120 kg/hm^(2),且在现蕾初期采摘叶片可获得较好收益。

饮料中甜菊糖苷水解条件的优化及高效液相色谱法测定

该文建立高效液相色谱(high performance liquid chromatography,HPLC)法测定饮料中甜菊醇的定量分析方法,并优化饮料中甜菊糖苷水解成甜菊醇的条件。采用3 mmol/g路易斯酸水解,无水乙醇提取,以70%乙腈水溶液(体积分数)作为流动相,使用ZORBAX SB-C18色谱柱。结果表明,甜菊醇在5.0μg/mL~100.0μg/mL的浓度范围内线性关系较好(r2≥0.999),平均回收率在81.9%~92.3%,相对标准偏差在1.17%~3.67%。饮料中甜菊糖苷转化甜菊醇的最佳工艺条件:催化剂为氯化铁,提取温度100℃,甜菊糖苷浓度300 mg/mL,提取时间3 h,催化剂含量3 mmol/g。

甜菊糖苷在食品中的应用及发展趋势分析

随着社会不断发展,人们的生活质量不断提高,低热量高甜度的天然甜味剂成为越来越多人的选择。而甜菊糖苷作为一种从草本植物甜叶菊中提取出来的天然甜味剂,具有来源广泛、价格低廉、甜度高、热量低等特点,被广泛应用于饮料、甜品、酒类等食品中。本文对甜菊糖苷的研究现状进行分析,重点阐述了甜菊糖苷在食品中的应用,并对甜菊糖苷的发展前景进行了综述,皆在为甜菊糖苷的进一步发展提供理论支撑。

苹果酱低糖风味酸奶研制及贮藏品质评价

通过单因素实验和正交实验研究了甜菊糖苷替代蔗糖量、苹果酸添加量和苹果酱添加量对酸奶的感官评分影响,同时研究了苹果酱低糖酸奶在20 d贮藏期内酸度和乳清析出率的变化特点。结果表明:苹果酱低糖酸奶最适生产配方为甜菊糖苷替代蔗糖量30%、苹果酸0.06%、苹果酱10%,43℃发酵6 h,制成酸奶感官评分最高(92分),酸度77.8°T,产品表面光滑平整,组织细腻均匀,无乳清析出;苹果酱低糖酸奶在4℃时冷藏20 d后,随着冷藏时间的延长,产品的酸度和乳清析出率均呈现增加趋势,但均低于普通酸奶。该研究结果为果酱低糖酸奶的研究和生产提供参考。

甜菊糖苷的特性及应用

甜菊糖苷作为一种甜味剂正在不断的被认同。文章简要的介绍了甜菊糖苷的化学性质和化学结构、理化性质、分离纯化及检测方法。综述了甜菊糖苷在药理保健方面的作用以及在食品工业、医药业和化工方面的应用,并预测甜菊糖苷将成为一种很有发展前景的甜味剂。

大孔树脂D392对莱鲍迪苷A和甜菊苷吸附作用的研究

根据莱鲍迪苷A(RA)比甜菊苷(S)多一个葡萄糖基的特点,针对性地选择了14种工业树脂,分别考察了它们对RA和S的吸附能力和吸附选择性。在此基础上,研究了吸附选择性较高的大孔树脂D392的吸附动力学和吸附热力学,并考察了温度、甜菊糖苷水溶液浓度、pH、溶剂对D392吸附选择性的影响规律。结果表明,D392在水相中对S的吸附能力大于对RA的吸附能力,吸附为放热过程,且在较低温度下吸附选择性较好。在298.15K,pH7.0,混合糖苷(RA/S=1∶1)浓度为5g/L时,经D392吸附6h,吸附残液中RA/S达到最大值。进一步以RA含量70.3%的甜菊糖苷作供试液,经D392吸附6h,吸附残液中RA含量可提高到88.4%,RA保留率为68.0%。

甜叶菊糖苷的应用和安全性的研究进展

甜叶菊[stevia rebaudiana(bertoni)bertoni]原产南美巴拉圭高原南纬2526度之间的常绿小灌木;甜叶菊叶片的提取物含有8种甜叶菊糖苷;它们是一类四环二萜类衍生物,其中Rebaudioside A,stevi-oside分别是10%蔗糖溶液甜度的300倍和450倍,而且具有低发热量,对龋齿、高血压、糖尿病、肥胖症具有一定治疗作用,也适于苯丙酮尿症患者食用,所以一直受人们的关注。本文综述了甜叶菊糖苷在应用方面及其安全性的研究进展。

不同基因型甜叶菊光合生理特性与产量品质比较

为了筛选适宜甘肃河西绿洲灌区种植的甜叶菊及高光效育种材料,对引进的14个不同基因型甜叶菊种质资源光合生理特性、产量和甜菊醇糖苷含量进行差异性分析。结果表明,不同基因型甜叶菊整个生育期光合速率(photosynthetic rate,Pn)、蒸腾速率(transpiration rate,Tr)、气孔导度(stomata conductance,Gs)变化均呈"低-高-低"的单峰曲线,且峰值都出现在现蕾期。各次测定表明各基因型间Pn、Tr和Gs与干叶产量、甜菊醇糖苷含量具有一定的相关性。HX-2和ZY-0911两个基因型光合速率和干叶产量均较高,分别较平均值提高9.18%、8.66%和6.41%、9.68%。基因型间甜菊醇糖苷含量差异达到极显著水平(P<0.01),BY-0915-2和ZY-0911甜菊醇糖苷含量最高,分别为15.33%和14.30%,较平均增加31.31%和22.49%。综合分析得出,甘肃河西绿洲灌区要引种高光效甜叶菊进行栽培或作为育种亲本时,应首选引自安徽的8(ZY-0911)。

甜菊醇糖苷生物合成途径关键酶基因KA13H的克隆及序列分析

本研究利用RT-PCR方法从甜叶菊叶片中分离了一个与甜菊醇糖苷生物合成密切相关的基因KA13H,对该基因的ORF、编码产物结构、同源性比对及次级结构等进行了生物信息学预测分析,同时初步分析该基因的组织表达和原核表达。经DNAMAN6.0软件分析,该基因编码一条分子量为54.476kD的由476个氨基酸残基组成的多肽,含有典型的细胞色素P450的血红素结合位点FXXGXXXCXG,TMPRED程序预测其C-端含有一个明显的跨膜区MIQVLTPILLFLIFFVFWKVY,是一个典型的细胞色素P450基因。推断的KA13H编码产物与其它生物合成相关细胞色素P450同源比对和系统发生分析表明,该蛋白与苜蓿(Medicago truncatula)CYP716A12和云杉(Sitka spruce)CYP720B1的一致性较高,分别为51%和46%,推测KA13H可能与CYP716A12和CYP720B1具有类似的功能。KA13H次级结构分析结果表明,KA13H与CYP74A2的一致性仅为15%,但是它们却有着类似的次级结构,都含有6个基质识别位点(SRSs)。半定量RT-PCR分析表明:KA13H在根、茎、叶和花中呈组成型表达,叶和花中的表达丰度较高;将该基因融合到原核表达载体pGEX-4T-1中,在原核中得到了成功表达。根据本研究结果,我们推测KA13H可能在甜菊醇糖苷生物合成过程中发挥着重要作用,该基因的克隆和表达分析为进一步深入了解甜菊醇的生物合成过程中相关酶和基因的功能奠定了基础。

甜菊糖苷的超声波提取及纯化工艺研究

研究了甜菊糖苷的超声波提取和纯化工艺。其中,甜菊糖苷的超声波提取的最佳工艺:在超声功率为200W条件下,以水作为提取剂,料液比1∶8,提取温度80℃,提取时间60min,甜菊糖苷的提取率为12.78%,较传统方法高10.27%。进而对提取液用醇沉法除杂,AB-8树脂富集,70%乙醇洗脱,活性氧化铝脱色,再经氧化铝柱层析深度脱色后,真空干燥得白色粉末,纯度为92.15%。该工艺具有用时短,提取率高,成本低,产品纯度高以及工艺绿色环保的优点。

高效液相色谱法测定甜叶菊叶片中甜菊糖苷含量

为检测化学调控下甜叶菊叶片中不同甜菊糖苷的含量,建立了快速分析甜叶菊叶片中3种甜菊糖苷含量的高效液相色谱测定方法。液相色谱分析采用Sepax HP-Amino(4.6×250 mm,5μm)色谱柱,柱温40℃,流速1.00 mL/min,流动相采用等度洗脱模式,流动相乙腈/水组成比为80∶20(v/v),检测器为紫外-可见检测器,检测波长210 nm,谱带宽度20 nm,狭缝宽度16 nm,进样体积4μL;样品采用超声波辅助热水提取,澄清石灰水去除杂质。结果表明液相色谱分析得出3种甜菊糖苷(甜菊糖苷、莱鲍迪苷C、莱鲍迪苷A)线性方程分别为Y=1.075X-1.853(r=0.999 9)、Y=1.302 X+1.136(r=0.999 5)和Y=1.117X+1.089(r=0.999 8),在0.05～5.00 mg/mL之间线性关系均表现良好,方法检测限分别为1.091μg/mL,3.062μg/mL和1.078μg/mL。所建方法简单灵敏,适合叶片样品快速检测。