Optimization of Glycoalkaloid Analysis for Use in Industrial Potato Fruit Juice Downstreaming

Annually, within the European Union about 1.7 million tons of starch is produced by processing over 8 million tons of potato tubers, Solanum tuberosum. In recent years, the potato protein content has gained tremendous industrial interest, since these proteins have excellent nutritional value. As naturally occurring, secondary plant metabolites steroidal potato glycoalkaloids are formed in potatoes. The two major glycoalkaloids in potatoes are α‐solanine and α‐chaconine. Because of the significant toxicity of the glycoalkaloids for human and for animal nutrition it was essential to develop efficient extraction processes. The need for an easy, fast, sensitive and reliable glycoalkaloid assay at the very beginning of the production chain is obvious. In this study an efficient analytical assay for potato glycoalkaloids from powdery protein samples under industrially relevant conditions is described: sample extraction, analyte pre‐purification, and final HPLC analysis. An acetic acid extraction/homogenization process was used for glycoalkaloid extraction from potato protein samples. The extracts were purified by means of solid phase extraction cartridges using the different washing steps developed in this study. The final determination was performed through an HPLC method using a Reprosil‐Pur NH₂ column. The limit of detection was 5 μg/mL for α‐solanine and α‐chaconine, respectively, corresponding to concentrations of 20 ppm in potato protein powder.     

High-level expression of soluble human β-defensin-2 in Escherichia coli

Human β-defensin-2 (hBD2) is a short cationic peptide with a broad antimicrobial spectrum. The coding sequence of hBD2 was cloned into pET-32a (+) to construct a fusion expression plasmid, pET32–hBD2, which was transformed into E. coli BL21 (DE3) for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the fusion protein in soluble form efficiently and to avoid the formation of insoluble inclusion bodies. The optimal conditions were determined as following: cultivation at 28 °C in MBL medium, induction at middle stage of exponential growth with 0.8 mM IPTG, and post-induction expression for 8 h. Under the above conditions, a high percentage of the target fusion protein (≥92.3%) was expressed in soluble form and the volumetric productivity of soluble fusion protein reached 1.3 g/l. The culture process was successfully scaled up in a 10 l bench-top fermentor.     

重组大肠杆菌产Streptomyces sp. FA1来源木聚糖酶的摇瓶发酵优化

为了实现来源于Streptomyces sp. FA1的木聚糖酶的高效胞外分泌表达,对E.coli BL21(DE3)/pET20b(+)/coe/xynA基因工程菌的发酵产酶诱导条件进行优化,获得最优的诱导条件为25 ℃发酵6 h后添加终浓度为0.4 mmol/L的IPTG。在此基础上对发酵培养基进一步优化,得到最优培养基成分为:甘油11 g/L,酵母粉24 g/L,蛋白胨8 g/L,磷酸盐浓度89 mmol/L,镁离子4 mmol/L。最终酶活达到780.2 U/ml,为未优化前的2.2倍,是目前大肠杆菌摇瓶发酵产木聚糖酶的最高表达水平,为实现该酶的工业化生产奠定基础。     

益生菌Lactobacillus casei Zhang增殖培养基的优化

Lactobacillus casei Zhang是一株分离自传统酸马奶中的益生菌。本文研究了不同碳源、氮源、碳氮比例、微量元素及缓冲盐对Lactobacillus casei Zhang增殖培养的效果, 并采用响应面法对优选的碳源、氮源和缓冲盐类的组成含量进行优化, 得到Lactobacillus casei Zhang的增殖培养基为：葡萄糖 20.9 g/L、大豆蛋白胨10.45 g/L、酵母粉10.45 g/L、K2HPO4 3.5 g/L、醋酸钠14.6 g/L、柠檬酸钠2.3 g/L、MgSO4?7H2O 1 g/L、MnSO4?5H2O 54 mg/L、CuSO4?5H2O 10 mg/L、吐温80为1 g/L。Lactobacillus casei Zhang在此增殖培养基中经37℃18 h培养活菌数可达到4.78×109 CFU/mL, 比在MRS中(4.8×108 CFU/mL)提高近10倍。     

获取复配农药最佳增效配方的一种简易方法

借鉴共毒因子法评价农药复配联合作用的思想,将参与复配的单剂引起25％死亡率的剂量定为零水平,利用二次回归通用旋转组合设计安排实验研究氯氰菊酯和喹硫磷复配对斜纹夜蛾（Spodoptera litura）2龄幼虫的增效作用,得出杀虫剂使用量与斜纹夜蛾幼虫死亡率机率值之间的关系,从共毒系数（CTC）的计算公式推导出共毒系数与杀虫混剂中两单剂使用浓度N1、N2之间的数学关系,CTC＝100／N1／α N2/b（α、b分别为氯氰菊酯和喹硫磷的LC50）,进而确定最优化问题的目标函数j=N1/α＋N2／b,利用国际统计分析软件SAS的NLP过程求最大共毒系数和最优配方,所得结论与共毒系数法一致。     

苦丁茶冬青的RAPD影响因素及实验条件的优化

以苦丁茶冬青为材料研究随机扩增多态DNA(RAPD)的影响因素及各种实验条件优化。研究结果表明：模板DNA的浓度适宜范围为20ng／反应-80ng／反应RAPD均可得到一致的结果;dNTVs的适宜浓度范围为200μmol／L-400μmol／L;Mg^2 适宜浓度范围为1．5mmol／L-2．0mmol／L;其合适的复性温度为35—37℃;2min的延伸时间,45次热循环。按照此优化的RAPD条件进行重复实验,实验结果重现性良好,因而确定了苦丁茶冬青RAPD反应体系之最佳的实验条件。     

A reduced muscle model and planar musculoskeletal model fit for the simulation of whole-body movements

Musculoskeletal models are made to reflect the capacities of the human body in general, and often a specific subject in particular. It remains challenging to both model the musculoskeletal system and then fit the modelled muscles to a specific human subject. We present a reduced muscle model, a planar musculoskeletal model, and a fitting method that can be used to find a feasible set of active and passive muscle parameters for a specific subject. At a minimum, the fitting method requires inverse dynamics data of the subject, a scalar estimate of the peak activation reached during the movement, and a plausible initial estimate for the strength and flexibility of that subject. While additional data can be used to result in a more accurate fit, this data is not required for the method solve for a feasible fit. The minimal input requirements of the proposed fitting method make it well suited for subjects who cannot undergo a maximum voluntary contraction trial, or for whom recording electromyographic data is not possible. To evaluate the model and fitting method we adjust the musculoskeletal model so that it can perform an experimentally recorded stoop-lift of a 15 kg box.     

Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO₂ reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO₂ reduction.     

Development and evaluation of different strategies for the clean synthesis of silver nanoparticles using Yarrowia lipolytica and their antibacterial activity

An environment-friendly, cheap method, biogenic synthesis of silver nanoparticles (AgNPs) is interesting as compared to physical and chemical synthesis methods. The aim of the present study was to utilize the inherent capability of Yarrowia lipolytica as a novel biocatalyst for green production of AgNPs using different strategies, including growing cells, resting cells, and cell-free extracts (CFE) under optimized reaction conditions. The produced AgNPs were evaluated with UV–vis spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, and Fourier transform infrared spectrometry. In the growing cells strategy, Y. lipolytica produced spherical AgNPs under the optimized conditions, 2.5 mM of silver ions, 7.5 g/l of yeast biomass, a temperature of 30 °C, a pH of 6, and a shaking rate of 50 rpm after 48 h. The sizes and monodispersity of the AgNPs in the resting cells strategy were better than those in the other two. However, the AgNPs were produced faster in the CFE strategy. The antibacterial activity and minimal inhibitory concentration of the AgNPs against certain Gram-positive and Gram-negative bacteria were determined by the agar well diffusion and broth microdilution methods. The AgNPs had a considerable antibacterial effect compared to chloramphenicol as a broad-spectrum antibiotic.     

An EMG-CT method using multiple surface electrodes in the forearm

Electromyography computed tomography (EMG-CT) method is proposed for visualizing the individual muscle activities in the human forearm. An EMG conduction model was formulated for reverse-estimation of muscle activities using EMG signals obtained with multi surface electrodes. The optimization process was calculated using sequential quadratic programming by comparing the estimated EMG values from the model with the measured values. The individual muscle activities in the deep region were estimated and used to produce an EMG tomographic image. For validation of the method, isometric contractions of finger muscles were examined for three subjects, applying a flexion load (4.9, 7.4 and 9.8 N) to the proximal interphalangeal joint of the middle finger. EMG signals in the forearm were recorded during the tasks using multiple surface electrodes, which were bound around the subject’s forearm. The EMG-CT method illustrates the distribution of muscle activities within the forearm. The change in amplitude and area of activated muscles can be observed. The normalized muscle activities of all three subjects appear to increase monotonically with increases in the load. Kinesiologically, this method was able to estimate individual muscle activation values and could provide a novel tool for studying hand function and development of an examination for evaluating rehabilitation.