The Use of Biopreservatives in the Control of Bacterial Contaminants of Sugarcane Alcohol Fermentation

ABSTRACT: Efficacy and safety of biopreservatives were studied for microbiological control of alcohol fermentation. Minimum inhibitory concentrations of nisin and lysozyme, and combinations with EDTA and Tween 20, were determined for Lactobacillus plantarum, L. fermentum , and L. confusus. The highest MIC value for nisin was 9.0 ppm for L. fermentum. Lysozyme was efficient against L. plantarum (700 ppm). A 2⁴ factorial design was used to study the effect of biopreservatives and potentiators on the growth kinetics of Lactobacillus fermentum. The combination of 8.6 ppm of nisin plus 0.1% of Tween 20 delayed the lag phase by 12 h. The biopreservatives did not affect the fermentation kinetics of Saccharomyces cerevisiae.     

剩余污泥资源化提取蛋白质方法的优选

剩余污泥中蛋白质的资源化利用是目前研究的热点,污泥预处理则是实现污泥中蛋白质释放的重要途径。为了进一步提高剩余污泥中蛋白质的溶出效果,选取热碱预处理、超声碱联合预处理、溶菌酶预处理对污泥进行溶胞,以蛋白质提取浓度为主要指标进行参数优化,并利用等电点法对粗提取蛋白进行纯化回收。结果表明：溶胞效果热碱预处理（pH值13、温度140℃、时间1.5 h,2 062.98 mg/L）>超声碱联合预处理（497.76 mg/L）>溶菌酶预处理（269.95 mg/L）,且在pH值为3时热碱预处理蛋白质纯化回收率可达62.42%。试验结果表明：热碱预处理在提取效果方面较另外两种方法优势明显,具有良好的利用前景。     

A molecular dynamics simulation of bacteriophage T4 lysozyme

An analysis of a 400 ps molecular dynamics simulation of the164 amino acid enzyme T4 lysozyme is presented. The simulationwas carried out with all hydrogen atoms modeled explicitly,the inclusion of all 152 crystallographic waters and at a temperatureof 300 K. Temporal analysis of the trajectory versus energy,hydrogen bond stability, r.m.s. deviation from the startingcrystal structure and radius of gyration, demonstrates thatthe simulation was both stable and representative of the averageexperimental structure. Average structural properties were calculatedfrom the enzyme trajectory and compared with the crystal structure.The mean value of the C displacements of the average simulatedstructure from the X-ray structure was 1.1 ± 0.1 Å;differences of the backbone and angles between the averagesimulated structure and the crystal structure were also examined.Thermal-B factors were calculated from the simulation for heavyand backbone atoms and both were in good agreement with experimentalvalues. Relationships between protein secondary structure elementsand internal motions were studied by examining the positionalfluctuations of individual helix, sheet and turn structures.The structural integrity in the secondary structure units waspreserved throughout the simulation; however, the A helix didshow some unusually high atomic fluctuations. The largest backboneatom r.m.s. fluctuations were found in non-secondary structureregions; similar results were observed for r.m.s. fluctuationsof non-secondary structure and angles. In general, the calculatedvalues of r.m.s. fluctuations were quite small for the secondarystructure elements. In contrast, surface loops and turns exhibitedmuch larger values, being able to sample larger regions of conformationalspace. The C difference distance matrix and super-positioninganalyses comparing the X-ray structure with the average dynamicsstructure suggest that a ‘hinge-bending’ motionoccurs between the N- and C-terminal domains.     

运动发酵单胞原生质球的形成和再生

运动发酵单胞(Zymomonas mobilis)ATCC29191菌株在含1～3g/L甘氨酸的液体培育12小时的菌体,用含20g/L的溶菌酶溶液处理12小时,可以稳定得到80％～90％的原生质球。原生质球稀释后在底层再生培养基上涂布,上面复盖一层半固体培养基,30℃培养5～7小时,再生率可达10~(-2)水平。     

分子动力学模拟HEWL晶体在不同环境中的动力学行为

简要阐述分子动力学模拟的原理及步骤,介绍研究溶菌酶的一般方法和优缺点。在Ubuntu操作系统环境下,利用Gromacs软件和其自带的Gromos96力场,通过分子动力学模拟(MD)鸡蛋清溶菌酶晶体(chicken egg-whitelysozyme,HEWL)溶液,考察真空、水溶液和加入NaCl 3种不同环境条件对溶菌酶晶体构象动力学行为的影响,发现无论从均方根位移(rmsd)、回旋半径、还是从B因子值的轨迹图分析,HEWL在水溶液特别是加入抗衡离子(Na~+,Cl~-)的水溶液的环境下的结构更稳定、合理,与(protein data bank)数据库的真实情况相符。原因是Cl~-与溶菌酶晶体在界面处发生了吸附现象,局部形成溶菌酶-Cl~-复合物,抑制了蛋白-水合物中水分子在相邻水合位置间的跳跃,从而使单晶体在离子液态中更加稳定。模拟结果表明,在pH值6.5,等电位点13.1,总电荷7.999 6的体系下,影响HEWL的吸附位点为123号残基(色氨酸),对从分子水平上解释HEWL晶体的动力学吸附行为具有重要指导意义。     

Influences of copolymers (Copovidone,Eudragit RL PO and Kollicoat MAE 30 DP) on stability and bioactivity of spray-dried and freeze-dried lysozyme

Protein stability is the most crucial factor in protein pharmaceutical preparations. Various techniques were applied for producing stable protein formulations such as spray-drying and freeze-drying. However, heating and freezing stresses are disadvantages for proteins using these methods, respectively. Accordingly, excipients have been used to preserve therapeutic effects of proteins during processing and for long period of time. Therefore, influences of Copovidone, Eudragit^® RL-PO and Kollicoat^® MAE-30 DP (as excipients) on stability and integrity of lysozyme (as a model protein) in spray-dried and freeze-dried forms were investigated. Protein formulations in both dried forms were prepared without and with the addition of mentioned excipients at different concentrations. Protein formulations were characterized for yield determination, morphology using scanning electron microscopic (SEM), thermal analysis by Differential Scanning Calorimetry (DSC), secondary structure stability using Fourier transform infrared (FT-IR) spectroscopy and biological activity. All protein formulations were subjected to a stability study as solid protein formulations for 3 weeks at 24?°C/76% relative humidity and aqueous protein samples were stored at 50?°C for 30?min in a water bath. Results showed that Copovidone successfully preserved integrity and biological activity of lysozyme before and after storage in both spray-dried and freeze-dried forms with more advantage for using higher concentration of the same excipient. Smooth spheres of spray-dried lysozyme formulations with Copovidone were smaller than spray-dried lysozyme without and with Kollicoat^® MAE-30 DP, which affected %yield produced. Copovidone has demonstrated valuable protection ability for lysozyme.     

Improvement of lysozyme recovery method from the precipitate of protein-anionic surfactant complexes

A new protein separation process using a surfactant and a polar organic solvent consists of a precipitation step and a recovery step. In the precipitation step, a protein-surfactant complex is precipitated from an aqueous solution, when an ionic surfactant, sodium di(2-ethylhexyl) sulfosuccinate (AOT), is added to an aqueous solution, including protein (lysozyme). In the recovery step, the precipitate is dissolved in a polar organic solvent, such as acetone, and the protein is recovered as precipitates when a very small amount of salt solution was added to remove surfactants from the protein-surfactant complex. However, the details of the protein recovery step from precipitate have not been studied yet. In this study, the improvement of the protein recovery step was examined from the viewpoint of a recovery ratio of protein and a remaining ratio of surfactant. The optimum NaCl concentration in the feed for the protein recovery was in the range of 0.05–0.2 kmol/m³. As the NaCl concentration in the feed increased to more than 0.2 kmol/m³, the precipitation ratio decreased due to the electrostatic screening effect of NaCl. It was found that the addition of a very small amount of NaCl solution to acetone was unnecessary when NaCl was included in the feed lysozyme solution. On the other hand, as the NaCl concentration decreased to less than 0.05 kmol/m³, the precipitation ratio was decreased due to the low re-precipitation of protein by the addition of a small amount of NaCl solution in acetone. In the case of the feed containing no salt, the desired NaCl concentration added to acetone was in the range above 0.2 kmol/m³. In addition, the most suitable volume ratio of acetone to feed was found to be 0.2.     

Evaluation of the effects of sodium chloride,potassium sorbate,nisin and lysozyme on the probability of growth of Clostridium sporogenes

The aim of this study was to evaluate the combined effect of salt (sodium chloride, 0–8% w/v), sorbate (potassium sorbate, 0–4.5% w/v), nisin (0–500 ppm) and lysozyme (0–500 ppm) on the survival of Clostridium sporogenes as a non‐toxigenic surrogate of Clostridium botulinum in terms of the probability of growth by using a central composite rotatable design. The results indicated that salt and sorbate were the most effective factors in preventing the growth of Cl. sporogenes in high‐moisture (>95%) and low‐acid conditions. The probability of growth of Cl. sporogenes in broth was reduced by combinations of salt and sorbate. Nisin and lysozyme had insignificant effects on the probability of growth of Cl. sporogenes (P > 0.05). Lysozyme individually and in combination with nisin had no inhibitory effect on Cl. sporogenes. Overall, the addition of sorbate and lysozyme may allow the salt concentration to be reduced while preventing growth.     

Electrospinning pure protein solutions in core–shell fibers

Electrospinning of protein‐loaded fibers faces many challenges, e.g. burst release owing to segregation of the protein on the fiber surface, loss of activity due to electrospinning conditions, limitation of loading capacity etc. Core–shell electrospinning provides an effective way to electrospin fibers wherein the core can be loaded with bioactive molecules in friendly conditions of a compatible polymer solution, thereby protecting the molecules from the electrostatic field and organic solvent of shell solutions. The shell polymer, after the electrospinning, acts as a barrier to control the release of the loaded molecules. However, the limitation of loading capacity still remains due the prerequisite of using an additional polymer as additive to achieve the minimum viscosity of the core solution required for viscous drag by the shell solution being drawn by the electrostatic force. The work reported here aims to alleviate the need of a polymer additive by using aqueous protein solutions of very high concentration. High concentrations of protein solutions were successfully electrospun as the core of the protein–poly(lactide‐co‐glycolic acid) core–shell fibers. A partitioning effect was seen in the controlled release of hydrophilic proteins as they were retained in the aqueous core for longer times. Using lysozyme as a model protein, it was shown that the activity is significantly retained after electrospinning, compared with electrospinning in monolithic fibers. Moreover, the lysozyme activity was also comparable with the lysozyme released from core–shell fibers spun using poly(vinyl acetate) as additive in the core. Copyright © 2012 Society of Chemical Industry