绿色低共熔溶剂提取互叶白千层精油及成分分析

为探究绿色低共熔溶剂提取互叶白千层精油的最佳工艺,该研究采用四种提取工艺作对比,并在单因素实验的基础上结合响应面进行了工艺优化,利用气相色谱-质谱法（GC-MS）对四种工艺提取到的互叶白千层精油进行了比较分析。结果表明：（1）互叶白千层精油最佳提取工艺为干燥原料低共熔溶剂蒸馏,最优提取条件为氯化胆碱与1,3-丁二醇摩尔比1∶3、含水量52 mol、原料干燥时间9 h、料液比1∶5 g·mL^-1、蒸馏时间60 min,在此条件下所得提取率为4.06%,实验值与响应面模型的预测值有较好的拟合性,证实模型有效,提取率比新鲜原料水蒸气蒸馏、干燥原料水蒸气蒸馏和冻融原料水蒸气蒸馏分别提高了383.33%、290.38%和497.06%。（2）经四种工艺提取所得精油主要成分大致相同,但含量差距很大,主要有醇类和烯烃类物质。（3）干燥原料低共熔溶剂蒸馏的精油提取率和特征组分含量都高于其他工艺,包括醇类8种,烯烃类9种,其他化合物3种;主要成分为γ-松油烯、松油烯、α-蒎烯、α-松油醇、香树烯,其中烯烃类化合物相对含量最大,为45.31%;特征组分松油烯-4-醇含量为30.58%...     

天然低共熔溶剂促进纤维素酶水解橄榄苦苷的研究

橄榄苦苷酶水解产物具有良好的生物活性,为了促进纤维素酶水解橄榄苦苷,本研究通过天然低共熔溶剂(natural deep eutectic solvent, NADES)对纤维素酶稳定性的促进作用,形成NADES-纤维素酶溶剂系统促进橄榄苦苷的酶水解。以橄榄苦苷酶水解率为指标,考察温度、pH和DES种类及浓度对纤维素酶水解橄榄苦苷的影响。结果表明：在50℃,pH=5时水解3 h,以10%(V/V)DES-5(甜菜碱∶1,4-丁二醇=1∶2,n/n)为溶剂系统的橄榄苦苷酶水解率是缓冲液的1.7倍;证明以氯化胆碱或甜菜碱为氢键受体(hydrogen bond acceptor, HBA),多元醇为氢键供体(hydrogen bond donor, HBD)的NADES对增强纤维素酶稳定性、促进橄榄苦苷酶水解具有良好的效果,为高效制备橄榄苦苷酶水解产物提供了科学依据。     

超声辅助低共熔溶剂提取沙棘籽粕多酚的工艺优化

以一系列低共熔溶剂为提取剂,采用超声波辅助法从沙棘籽粕中提取多酚。在单因素试验结果基础上,利用Box-Behnken实验设计,运用响应面分析法对影响沙棘籽粕多酚得率的主要因素（超声功率、超声时间、超声温度）进行优化。结果表明,沙棘籽粕多酚最佳提取工艺条件为：以含水量为30%的氯化胆碱-草酸低共熔溶剂为最佳提取剂,液料比为14∶1,超声功率420 W,超声时间56 min,超声温度44℃。在此条件下,多酚得率为3.31±0.008%。对比试验发现：氯化胆碱-草酸低共熔溶剂对沙棘籽粕多酚的得率明显优于传统溶剂;与热回流提取相比,超声提取法具有明显的优势。     

基于绿色低共熔溶剂法高效提取鸡骨草中的黄酮和皂苷

为了研究鸡骨草中总黄酮和总皂苷的最佳提取工艺,通过单因素试验和星点设计-响应面法,对低共熔溶剂的性质(如种类、组分摩尔比、含水量)、提取温度、提取时间、液料比等多个因素进行考察。结果显示,总黄酮的最佳提取条件为:摩尔比1∶2、含水量30%的氯化胆碱/乙二醇作溶剂,提取温度80℃,提取时间40min,液料比15∶1(mL/g);总皂苷的最佳提取条件为∶摩尔比1∶4、含水量25%的氯化胆碱/乳酸作溶剂,提取温度80℃,提取时间64min,液料比56∶1(mL/g)。在最佳条件下,总黄酮和总皂苷的提取率较传统提取溶剂分别提高了33.3%和96.4%。本研究为鸡骨草中黄酮和皂苷的高效、安全提取提供了一条新思路。     

低共熔溶剂对酶的影响及应用研究*

低共熔溶剂是由一定化学计量比的氢键受体和氢键供体组合而成的新型绿色溶剂,具有成本低、易制备、环境友好等特点,可以作为普通有机溶剂和离子液体的替代溶剂。酶作为生物催化剂时反应条件温和,对反应底物专一性高,并且具有极高的催化效率和反应速度。酶促反应通常发生在水溶液体系,但近年来发现在低共熔溶剂中酶促反应也能有效进行。综述酶与低共熔溶剂共同作用的机理以及低共熔溶剂在酶促反应中的应用,展望未来的研究方向,为酶促反应体系的进一步开发奠定理论基础。     

棘孢曲霉转化甜菊糖为甜菊醇及纯化莱鲍迪苷A

【目的】本工作对棘孢曲霉固体发酵抽提酶液转化甜菊糖进行了研究,并对转化产物进行鉴定及纯化分析。【方法】用高效液相色谱、液质联用及红外光谱等方法对转化新产物进行鉴定,对上清液中莱鲍迪苷A(RA)成分进行纯化。【结果】棘孢曲霉酶液在10 h内对甜菊糖中的甜菊苷(SS)、莱鲍迪苷C(RC)进行高效特异性转化,以沉淀的形式析出的转化产物经鉴定为甜菊醇,转化率高达98.0%,分离提纯后纯度为95.2%,回收率达84.0%.由于甜菊醇的沉淀分离,留在溶液中的RA更易被纯化。RA通过树脂吸附分离的回收率为80.5%.【结论】棘孢曲霉酶液对甜菊糖的一次转化可以同时得到甜菊醇和莱鲍迪苷A两种产品,是一种经济高效的工艺。     

低共熔溶剂预处理对笋壳木质素结构及物化性质的影响

以慈竹(Neosinocalamus affinis)笋壳为原料,采用氯化胆碱和乳酸低共熔溶剂预处理提取氯化胆碱-乳酸(choline chloride-lactic acid, CC-LA)木质素,并对木质素结构、热稳定性及抗氧化活性进行表征和分析。结果表明：CC-LA木质素的得率在37.67%～62.32%之间,远高于磨木木质素的得率(13.4%)。CC-LA木质素相对分子质量低,多分散性指数小于2,结构均一性好。在氯化胆碱与乳酸的物质的量之比为1∶3,处理温度为130℃条件下得到CC-LA₃130木质素,该木质素较其他CC-LA木质素和磨木木质素醇羟基含量最低,酚羟基含量最高,热稳定性最好,清除DPPH自由基活性最强,自由基清除指数为3.77,高于商业抗氧化剂2,6-二叔丁基对甲酚(2.11),本研究说明了氯化胆碱/乳酸在最佳条件下(1∶3,130℃)提取的木质素具有应用于抗氧化剂领域的潜力。     

芝麻渣中蛋白提取方法的比较研究

芝麻渣是芝麻榨油后的主要副产物,含有丰富的蛋白质.但是目前除了少部分被当成饲料或肥料使用外,大部分芝麻渣被作为废弃物丢弃,造成环境污染和蛋白资源的严重浪费.将芝麻渣中的蛋白质提取进行高值化利用,具有广阔的应用前景和重要的现实意义.以芝麻渣为材料,分别利用低共熔溶剂(deep eutectic solvent,DES)法...     

深共熔溶剂提取杨树桑黄多酚类物质工艺优化及提取物抗氧化活性研究

桑黄是一类应用广泛的药用真菌,桑黄多酚具有抗氧化、抗炎和降糖作用。本研究中的杨树桑黄属于桑黄中重要且能栽培的一种。本研究采用深共熔溶剂(deep eutectic solvent,DES)从栽培杨树桑黄子实体中提取多酚类化合物,考察了不同提取条件对提取率的影响。采用氯化胆碱与尿素组成的DES体系对多酚进行提取,并进一步采用响应面法对提取条件进行优化,获得最佳提取工艺参数为21 min、80 ℃、料液比1:260 g/mL。在此条件下,多酚的提取率高达(23.17±0.88) mg/g,远高于传统方法(12.45±1.88) mg/g。最优条件提取的多酚显示了很强的DPPH和ABTS的清除能力。由此可见,采用DES法从杨树桑黄子实体中提取酚类化合物比传统方法更有效。     

乳酸菌天然质粒提取方法的优化

将前人报道的乳酸菌质粒提取方法与大肠杆菌质粒提取方法相结合,对常用试剂盒质粒提取方法进行改进,建立了一种快速、安全、高效的乳酸菌质粒提取方法。提取过程中,溶菌酶最佳浓度为20mg/ml,最佳处理时间为30min,同时避免了毒性物质溴化乙锭的使用。多次实验结果表明,采用改进后的方法可明显提高乳酸菌天然质粒的提取效果。且重复性好,为下一步乳酸菌的分子改良奠定了基础。     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant-based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern-day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.     

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。     

Highly Oxygenated Labdane Diterpenoids from Stevia rebaudiana and Their Anti-Atherosclerosis Activities

Five unknown labdane diterpenoids Stevelins A–E ( 1–5 ), three known labdane diterpenoids ( 6–8 ) and three labdane norditerpenoids ( 9–11 ) were isolated from the Stevia rebaudiana. The structures were determined primarily via NMR spectroscopic data and HR-ESI-MS experiments. X-ray crystallography using CuK_α radiation was used to determine the absolute configurations of 1 , and the absolute configurations of 2–5 were deduced by electronic circular dichroism (ECD) calculations. The potential anti-atherosclerosis activities of all compounds were evaluated by measuring their inhibitory effects on the macrophage foam cell formation. As a result, most isolated compounds could significantly inhibit oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation, which suggests that these compounds may be promising candidates in the treatment for atherosclerosis.     

甜菊含甙量的变异及R－A型良种的选育

从甜味成分种类及含量的不同,研究甜菊（ＳｔｅｖｉａｒｅｂａｕｄｉａｎａＢｅｒｔｏｎｉ）实生群体和单株无性系的变化,从中选育优质甜味成分含量高的良种。主要结果：（１）在实生群体中,丰产株型（圆纺锤形株）占７．３％,其中,优质甜味成分Ｒ－Ａ含量超过Ｓｔ含量的Ｒ－Ａ型株占１０．９６％,它们的Ｒ－Ａ含量变幅为３．３～１２．０％。（２）Ｒ－Ａ型良种Ｊ－２单株无性系在繁殖达２０００万株时,叶片大小和含甙量均存在极显著差异,Ｒ－Ａ含量变幅为４．５～１２．２％。（３）从Ｒ－Ａ含量为３．８６％的实生群体中选出Ｒ－Ａ含量为７．０４～１２．０３％的单株及从Ｒ－Ａ含量为９．１０％的良种单株无性系中选出Ｒ－Ａ含量为１０．１５～１２．１５％的单株,它们的Ｒ－Ａ含量均大幅度提高。     

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.     

The Fertilization and Early Development of Embryo and Endosperm in Stevia rebaudiana Bertani

The mature embryo sac is surrounded by endothelium tapetum. It is composed or an egg apparatus, one central cell with secondary nucleus, and 1–6 antipodal cells. About the 6th hour after pollination, female and male nuclei fuse with each other. The syngamy occurred almost simultaneously with the fusion of an other sperm nucleus and the secondary nucleus, but the velocity of the latter is faster than that of the syngamy. The fertilization of Stevia rebaudiana Bertani belongs to the premitotic type. About the 8th hour after pollination, primary endosperm nucleus is in mitosis, its dividing orientation may parallel or at right angle to the long axis of the embryo sac, and gives rise to two initial endosperm cells. The first five divisions of the endosperm cells are of synchronism. At the stage of heart-shaped embryo, the endosperm cells show the signs of digestion and absorbed. The endosperm development is of the cellular type. About the 10th hour after pollination, zygote divides for the first time. The division of the zygote is always transverse. The embryo development conforms to the Asterad type.     

Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth,stevioside, NPK,and chlorophyll content of Stevia rebaudiana

Stevia rebaudiana Bertoni is a unique medicinal plant which is mostly utilized as a sugar substitute for diabetic patients. In this research, regenerated plantlets of stevia in tissue culture is transferred to pots in greenhouse and inoculated with plant-growth-promoting rhizobacteria (PGPRs) (Bacillus polymixa, Pseudomonas putida, and Azotobacter chroococcum) and arbuscular mycorrhizal fungus (AMF) (Glomus intraradices). The results showed that in comparison to control, inoculation with a single microorganism, significantly increased root and shoot biomass as well as stevioside, chlorophyll, and NPK content in plants. However, such increased effects have been found to be further enhanced significantly due to dual compatible mixtures of inoculants resulting from their strong synergistic relationships among themselves. All growth parameters recorded the highest in 60-days-old plants in the treatment of Glomus+Azotobacter and followed with Glomus+Bacillus and Azotobacter+Pseudomonas treatments, respectively. Furthermore, high-performance liquid chromatography (HPLC) chromatograms revealed that the highest stevioside content have been produced in same treatments. Triple treatments had less positive effects compared to dual inoculations. Probably competence between microorganisms in triple inoculations has reduced their efficiency. Thus, suitable combination of mycorrhizal fungi and PGPR as biotic elicitors can enhance growth and stevioside content in tissue culture-regenerated plantlets of stevia.     

Plasmalemma Invaginations in Cultured Callus of Stevia rebaudiana: Ultrastructure and Ultracytochemical Localization of Acid Phosphatase

The plasmalemma of cells within meristematic regions was observed to possess invaginations in cultured callus of Stevia rebaudiana under differentiation. The ultrastructure and acid phosphatase (AcPase) ultracytochemistry Of these invaginations were studied. The plasmalemma invaginations occurred in the cells at various stages of vacuolation. In cells with dense protoplasm, plasmalemma appeared undulated but occasionally spherical and variable in size with conspicuous invaginations that projected into the peripheral cytoplasm. In the partially vacuolated cells, plasmalemma invagination became voluminously enlarged with increased contents and structurally complexed. In vacuolated cells, the enlarged invaginations protruded into the central vacuole but were delimitted from the tonoplast by an intermembrane zone continuous with the peripheral cytoplasm. Complex accumulations of membranes consisting of vesicular and coiled membranous Structures might develop within the plasmalemma invaginations. AcPase localization demonstrated high enzymic activity in the plasmalemma and its associated invagination. It seemed likely that these invaginations were functionally analogous to the vacuoles and therefore constituted part of the lytic compartment in these cells.