以4-香豆酸为底物转化白藜芦醇大肠杆菌工程菌的构建

白藜芦醇具有多种生物活性,在食品、化妆品及生物制药中具有广阔的应用前景。利用合成生物学生产白藜芦醇是未来的发展趋势。将白藜芦醇合酶(resveratrol synthase,RS)和4-香豆酰-CoA连接酶(4-coumaric acid-CoA ligase,4CL)的编码基因整合到共表达载体pETDuet-1上成功构建了重组质粒pETDuet-rs-4cl,转化大肠杆菌BL21(DE3),在M9培养基中添加4-香豆酸,在诱导条件下培养,发酵液中白藜芦醇含量为8.6 mg/L。本研究在大肠杆菌中实现了白藜芦醇的转化,为微生物法生产白藜芦醇奠定了基础。     

微生物合成白藜芦醇的研究进展

白藜芦醇是植物源的多酚化合物,具有清除自由基、抗氧化、延长寿命等生理活性,在医药、保健品、化妆品等方面有着广阔的应用前景。目前,白藜芦醇主要采用植物提取的方法,对自然植物资源依赖严重,而且受植物成分含量和提取效率低的限制。近年来,合成生物学的迅速发展已使人们将注意力转向利用微生物合成白藜芦醇。通过在微生物中转入外源基因,成功构建出了白藜芦醇工程菌株,并从基因序列、组合方式及发酵工艺等方面进行了优化改造。本文综述了微生物合成白藜芦醇的研究进展。     

超声辅助萃取葡萄分子物白藜芦醇及其对动物运动能力的影响

本研究以葡萄果茎为主要研究对象,探索了一种通过超声辅助从葡萄中萃取白藜芦醇的先进萃取工艺。同时,通过构建萃取和降解白藜芦醇过程的反应速率常数一级反应动力学模型,研究了萃取时间与回收率、可溶性白藜芦醇、绑定白藜芦醇及降解化合物等组分含量之间的关系,并分析了获得了最佳萃取时间、最大回收率及干物质的量。研究结果显示:与传统乙醇/水溶剂萃取法相比,超声波辅助法萃取白藜芦醇的回收率提高了24%～30%。模拟结果表明:萃取时间对萃取过程中各组分的含量有着显著的影响。当萃取时间为3.48 min时,巨峰葡萄果实茎中白藜芦醇的含量为489.4μg/g干物质,此时回收率最大为79.8%;同时萃取过程中降解化合物含量为83.1μg/g干物质,降解物含量为13.6%。另一方面,赤霞珠葡萄果茎中白藜芦醇的最大回收率和降解化合物的含量分别占总白藜芦醇的80.6%和13.1%,其含量分别为194.8μg/g干物质和31.6μg/g干物质。此外,本研究研究了白藜芦醇对大强度运动大鼠心肌抗氧化能力的影响,结果表明大强度运动后,补充RES可以减少因运动所造成的心肌组织自由基损伤现象。     

白黎芦醇和香青兰总黄酮抑制哮喘小鼠TNF-α和IL-6的释放

为研究白藜芦醇和香青兰总黄酮对哮喘小鼠肿瘤坏死因子-α(TNF-α)和白细胞介素-6 (IL-6)水平的影响,本研究采用卵清蛋白致敏来建立小鼠哮喘模型,给予白藜芦醇和香青兰总黄酮干预后,采用ELISA法检测小鼠BALF中TNF-α和IL-6的含量。结果显示,95 mg·kg^-1·d^-1的白藜芦醇和香青兰总黄酮均可降低哮喘小鼠BALF中的TNF-α和IL-6;95 mg·kg^-1·d^-1的白藜芦醇和香青兰总黄酮对小鼠体重有影响。本研究表明,白藜芦醇和香青兰总黄酮对哮喘炎症因子TNF-α和IL-6的释放具有一定抑制作用。     

薄层层析-紫外分光光度法测定葡萄果皮中白藜芦醇及白藜芦醇苷

建立了葡萄果皮中反式白藜芦醇及反式白藜芦醇苷含量的薄层层析-紫外分光光度测定法.以甲醇提取目标组分,以高效硅胶GF254为固定相,甲苯:乙酸乙酯:甲酸(5:4:1)为展开剂,对反式白藜芦醇及反式白藜芦醇苷进行分离纯化.并用紫外分光光度法进行定量,波长为306 nm.结果表明,该方法测定反式白藜芦醇及反式白藜芦醇苷含量的线性关系良好,相关系数为0.9997;平均回收率为98.12%、97.23%;方法的精密度较高.并用该方法测定了酿酒红葡萄品种蛇龙珠不同生长时期的反式白藜芦醇和反式白藜芦醇苷含量.     

白藜芦醇合成酶基因在基因工程中的应用及功能研究进展

白藜芦醇合成酶(Resveratrol synthase,RS)是白藜芦醇(Resveratrol,Res)合成途径中的关键酶。以往研究报道,RS基因已在多种植物和微生物中进行了转化和表达,并在植物的代谢及调控等方面发挥生物学作用。文中主要围绕RS基因对植物的转化,及异源表达后植物体内代谢产物的变化,转RS基因对植物抗病原菌活性、抗自由基活性和生长发育的影响,以及利用RS基因在微生物中生产Res的相关进展进行了综述。并对RS基因在生物工程方面的应用前景进行展望。     

不同处理方法对灵芝β-葡聚糖降解效果的比较研究

本研究采用酸法、碱法、酶法和微波法对灵芝β-葡聚糖进行降解,通过降解率、产物分子量变化、产物聚合度分布等指标比较了不同方法的降解效果。结果表明,微波法降解率高达94%,处理后产物的分子量明显降低,寡糖产物聚合度分布广。酶法降解率约为40%,寡糖产物中含有DP2-5的成分。酸法及碱法降解率低于20%,寡糖产物少。研究表明,与其他3种方法相比,微波法降解率高、产物丰富、操作条件易于控制,是一种简单、高效的降解灵芝β-葡聚糖、制备灵芝β-葡寡糖的方法。     

白藜芦醇合酶的研究进展

白藜芦醇是一种重要的植物抗毒素,具有多种医疗保健作用,因此其应用前景 非常广泛,已引起多方关注。白藜芦醇合酶是白藜芦醇生物合成途径中的关键酶之一,它催化1分子4-香豆酰辅酶A和3分子丙二酰辅酶A反应合成白藜芦醇,它是白藜芦醇生物合成中惟一必需的酶,关于它的研究已广泛开展起来。本文综述了白藜芦醇的药理活性、白藜芦醇合 酶的酶学性质、诱导途径和机制以及分子生物学方面的研究进展。     

白藜芦醇在大鼠体内的吸收代谢分析

目的：高效液相色谱及气质联用法同时分析大鼠血浆和尿中白藜芦醇及白藜芦醇苷含量。方法：6w龄Wistar大鼠10只,适应性喂养1w后,分装于10个代谢笼中。实验前禁食10h,按体重灌胃给予大鼠50mg/kg白藜芦醇。高效液相色谱法检测大鼠血浆和尿中白藜芦醇及白藜芦醇苷的含量,并通过气质联用仪探索性分析不同时间血浆中代谢产物的种类。结果：白藜芦醇及白藜芦醇苷在0～10mg/L浓度范围内线性较好,其R2 分别为0.9995及0.999 6。白藜芦醇灌饲1h后血浆中白藜芦醇浓度达到最大值,约4.79μg/mL,而白藜芦醇苷则到3h后才达到峰值,且其浓度达到白藜芦醇原型含量的5倍,约23.78μg/mL。白藜芦醇及其糖基化衍生物可在白藜芦醇摄入后24h内经尿液大量排泄到体外,排泄率超过摄入量的一半,其中原型占90%以上。气质联用分析进一步发现,机体摄入白藜芦醇3h后血浆中代谢物种类及含量均达到峰值。结论：白藜芦醇进入大鼠体内后在血浆中主要以衍生物形式存在,且代谢主要在摄入后前3h内,摄入后24h内主要以原型形式经尿液排出。     

生物素化白藜芦醇的分离纯化与药效学评价

以DCC为催化剂,采用低温化学催化法合成生物素化白藜芦醇.产物采用Resourse RPC反相色谱柱分离、纯化,并且运用液质联用进行分析验证.MTT法测定产物体外对Hep G2、MCF-7肿瘤细胞的抑制作用.实验结果显示:通过HPLC-MS、紫外扫描分析鉴定证实,分离得到的产物确实是生物素化白藜芦醇,其保留了原有的体外抗肿瘤活性.此种化学合成方法反应条件温和,容易控制,操作方便.合成得到的白藜芦醇的生物素化衍生物,为下一步探讨白藜芦醇的结合蛋白提供简单有效的分子工具.     

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.     

Resveratrol affects in vitro rumen fermentation,methane production and prokaryotic community composition in a time- and diet-specific manner

This study aimed to investigate the effect of resveratrol on methane production, rumen fermentation and microbial composition under high-concentrate (HC) and high-forage (HF) diets using the in vitro fermentation system. A total of 25 mg of resveratrol was supplemented into 300 mg of either HC or HF diet. Methane production, total volatile fatty acid (VFA) concentration, molar proportion of VFA, metabolites of resveratrol and prokaryotic community composition were measured after 12 and 24 h of in vitro fermentation. Resveratrol reduced methane production (ml per mg of dry matter degraded) by 41% and 60% under both HC and HF diets (P < 0.001), respectively, and this result could be associated with the lower abundance of Methanobrevibacter (P < 0.001) in response to resveratrol. The molar proportion of propionate was significantly higher in the resveratrol group only under the HC diet (P = 0.045). The relative abundance of 10 bacterial genera was affected by the three-way interaction of treatment, diet and time (P < 0.05). Resveratrol was partly converted to dihydroresveratrol after 24 h of fermentation, and its degradation could be associated with microbes belonging to the order Coriobacteriales. Our results suggest that multiple factors (e.g. diet and time) should be considered in animal experiments to test the effect of polyphenol or other plant extracts on rumen fermentation, methane emission and microbial composition.     

Formulation and Optimization of Zinc-Pectinate Beads for the Controlled Delivery of Resveratrol

Preventive and therapeutic efficacies of resveratrol on several lower gastrointestinal (GI) diseases (e.g., colorectal cancer, colitis) are well documented. To overcome the problems due to its rapid absorption and metabolism at the upper GI tract, a delayed release formulation of resveratrol was designed to treat these lower GI diseases. The current study aimed to develop a delayed release formulation of resveratrol as multiparticulate pectinate beads by varying different formulation parameters. Zinc-pectinate (Zn-pectinate) beads exhibited better delayed drug release pattern than calcium-pectinate (Ca-pectinate) beads. The effects of the formulation parameters were investigated on shape, size, Zn content, moisture content, drug encapsulation efficiency, swelling–erosion, and resveratrol retention pattern of the formulated beads. Upon optimization of the formulation parameters in relative to the drug release profiles, the optimized beads were further subjected to morphological, chemical interaction, enzymatic degradation, and stability studies. Almost all prepared beads were spherical with ∼1 mm diameter and efficiently encapsulated resveratrol. The formulation parameters revealed great influence on resveratrol retention and swelling–erosion behavior. In most of the cases, the drug release data more appropriately fitted with zero-order equation. This study demonstrates that the optimized Zn-pectinate beads can encapsulate very high amount of resveratrol and can be used as delayed release formulation of resveratrol.     

Delivery of cytoplasmic proteins to autophagosomes

Autophagy represents a signaling-dependent regulated process that allows the degradation of some cellular proteins in autophagosomes, and plays a critical role in the management of cellular homeostasis under various stress conditions. In recent years, selective degradation of cytoplasmic proteins during stress has attracted considerable scientific interest. Here we examined the ability of resveratrol to induce autophagy in a variety of human cancer cell lines. We found that resveratrol-induced autophagy is accompanied by colocalization of proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) with the green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) in autophagosomes. In addition, we found that hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a previously shown PELP1-interacting protein, is co-recruited to autophagosomes in the presence of resveratrol. Although autophagy has been assumed to be a bulk and non-selective degradation process, in recent years, evidence of selective degradation of cytosolic proteins and organelles by autophagy is mounting. These observations suggest that the interaction of the target protein(s) with the delivery protein or proteins such as HRS facilitates the transport of certain cytoplasmic proteins to autophagosomes for their selective degradation, and thus, could influence the cytoplasmic as well as nuclear functions of nuclear receptor coregulators. Since PELP1 and, perhaps, other nuclear receptor coregulators are widely dysregulated in human cancers, these findings highlight the significance of the autophagic selective degradation of PELP1 following resveratrol (or other phytoestrogens) treatment in developing future strategies to use resveratrol under cancer prevention and therapeutic settings.     

On the stability of plasmid DNA vectors during cell culture and purification

Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4°C, >12 weeks at −20°C) prior to processing. With alkaline lysates, however, storage at −20°C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization. Authors Freitas and Azzoni contributed equally to this work.     

Production and Characterization of a Polymer from Arthrobacter sp

An Arthrobacter sp. isolated from a glucose-sucrose agar plate was found to produce a neutral, extremely viscous, opalescent extracellular polymer. Growth, polymer production, and rheological properties and chemical composition of the isolated polymer were examined. The polymer was found to be substantially different from other arthrobacter polymers. Some unusual properties included irreversible loss of viscosity with high temperature and degradation of the polymer during fermentation and upon storage at 4 degrees C. Other characteristics included dependence on sucrose for polymer production, relative pH stability, increased viscosity with increased salt concentration, and pseudoplasticity. The polymer was found to be composed primarily (if not entirely) of d-fructose. The fructose content and other characteristics suggested that the polymer was a levan.     

Effect of the fermentation pH on the storage stability of Lactobacillus rhamnosus preparations and suitability of in vitro analyses of cell physiological functions to predict it

Aims:  To investigate how cell physiological functions can predict the stability of freeze-dried probiotics. In addition, the effect of the fermentation pH on the stability of probiotics was investigated.
Methods and Results:  Fermenter-grown (pH 5·8 or 5·0) Lactobacillus rhamnosus cells were freeze-dried and their survival was evaluated during storage at 37°C, in apple juice and during acid [hydrochloric acid (HCl) and malic acid] and bile exposure. Cells grown at pH 5·0 were generally coping better with acid-stress than cells grown at pH 5·8. Cells were more sensitive to malic acid compared with HCl. Short-term stability results of Lact. rhamnosus cells in malic acid correlated well with the long-term stability results in apple juice, whereas the results of cell membrane integrity studies were in accordance with bile exposure results.
Conclusions:  Malic acid exposure can prove useful in evaluating the long-term stability of probiotic preparations in apple juice. Fermentation at reduced pH may ensure a better performance of Lact. rhamnosus cells during the subsequent acid-stress.
Significance and Impact of the Study:  The beneficial effect of lowered fermentation pH to Lact. rhamnosus stability during storage in apple juice and the usefulness of malic acid test in predicting the stability were shown.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Cyclodextrin-Based Nanosponges for Delivery of Resveratrol: <Emphasis Type="Italic">In Vitro</Emphasis> Characterisation,Stability, Cytotoxicity and Permeation Study

The aim of this work was to increase the solubility, stability and permeation of resveratrol by complexation with cyclodextrin-based nanosponges (NS). Nanosponges are recently developed hyper-cross-linked cyclodextrin polymers nanostructured to form three-dimensional networks; they are obtained by reacting cyclodextrin with a cross-linker such as carbonyldiimidazole. They have been used to increase the solubility and stability of poorly soluble actives. This study aimed at formulating complexes of resveratrol with β-cyclodextrin nanosponges in different weight ratios. DSC, FTIR and X-ray powder diffraction (XRPD) studies confirmed the interaction of resveratrol with NS. XRPD showed that the crystallinity of resveratrol decrease after encapsulation. The particle sizes of resveratrol-loaded NS are in between 400 to 500 nm with low polydispersity indices. Zeta potential is sufficiently high to obtain a stable colloidal nanosuspension. TEM measurement also revealed a particle size around 400 nm for NS complexes. The in vitro release and stability of resveratrol complex were increased compared with plain drug. Cytotoxic studies on HCPC-I cell showed that resveratrol formulations were more cytotoxic than plain resveratrol. The permeation study indicates that the resveratrol NS formulation showed good permeation in pigskin. The accumulation study in rabbit mucosa showed better accumulation of resveratrol NS formulation than plain drug. These results signify that resveratrol NS formulation can be used for buccal delivery and topical application.