膜材与制备过程对血红蛋白微胶囊粒径和包埋率的影响

以单甲氧基聚乙二醇聚乳酸共聚物（PELA）为膜材用复乳溶剂扩散法制备了包含牛血红蛋白（BHb）的微胶囊,微胶囊中BHb的P₅₀和Hill系数分别为3 466 Pa和2.4左右,接近于天然BHb的生物活性。研究发现膜材种类对BHb微胶囊包埋率和粒径的影响最大,使用MPEG2000为亲水性嵌段的PELA共聚物时,包埋率最高,达到90％以上,粒径为3～5 μm左右;随着膜材浓度的增大,微胶囊包埋率和粒径均增加;随着外水相NaCl浓度的增大,微胶囊包埋率升高、粒径减小;随着外水相稳定剂PVA浓度的增大,微胶囊粒径减小,包埋率先升高后降低,在较低浓度下（10 g/L、20 g/L）包埋率较高;初乳化搅拌速率的增大,有利于包埋率的提高,但对粒径影响不大;复乳化搅拌速率的影响较复杂,当复乳液体积较大时,复乳化搅拌速率对微胶囊制备的影响规律性不明显。当固定膜材和初乳化搅拌速率时,包埋率和粒径之间存在着类似抛物线的关系,包埋率随着粒径的减小而降低。     

拟除虫菊酯微胶囊的制备

以海藻酸钠、壳聚糖为壁材,氯氟氰菊酯为芯材,通过复凝聚法制备微胶囊。用红外光谱确定复合膜的形成,光学显微镜观察微胶囊的分散性和规整度,气相色谱法测定包封率。考察油水相体积比、搅拌速度、乳液稀释水用量及壳聚糖的浓度对氯氟氰菊酯微胶囊化的影响。结果表明,最佳的实验条件是油水相体积比为1∶10、搅拌速度为1300 r/min、乳液与水体积比为1∶1.2,壳聚糖浓度为0.1%。制备的微胶囊包封率可以达到80.3%。     

保加利亚乳杆菌微胶囊化工艺研究

目的制备保加利亚乳杆菌微胶囊,提高菌株的酸、热耐受性及降低菌体的分离成本。方法以保加利亚乳杆菌(Lactobacillus bulgaricus)为研究对象,海藻酸钠(SA)为壳材、CaCl2为固化剂,制备保加利亚乳杆菌微胶囊;包埋率、颗粒平均化程度、机械强度等为考核指标,研究保加利亚乳杆菌微胶囊化的工艺。结果当海藻酸钠浓度为0.75%、CaCl2浓度为3%、电压为600V、泵速为1.96mL/min、震动频率为80Hz时,微胶囊化包埋效果最佳,经固定化后的菌微胶囊保持了良好的保加利亚乳杆菌的活性,微囊化保加利亚乳杆菌经过2次连续发酵后的产酸量分别达到59.4g/L和55.8g/L。结论本研究为工业化生产乳酸提供了一条具有经济价值的途径。     

星点设计效应面法优化姜黄素微球的制备工艺

采用乳化溶剂扩散法来制备姜黄素微球。以包封率为评价指标,通过单因素试验法、星点设计效应面法优化制备处方工艺。最佳处方及工艺条件:乙基纤维素与姜黄素的质量比为20.04∶1,吐温80用量为21.68 mg/mL,转速为600 r/min,搅拌时间为30 min以及水相和油相体积比为1.47∶1。此条件下制备的姜黄素微球外形圆整,释放平稳,包封率高达93.66%,且制备工艺简单,效应面法建立的数学模型预测良好。     

艾叶油微胶囊的制备工艺研究

目的:研究艾叶油微胶囊的制备工艺。方法:采用复凝聚法制备艾叶油微胶囊,研究乳化时间、乳化转速、凝聚温度、凝聚转速等因素对艾叶油微胶囊成型性的影响,确定最佳制备工艺;采用热重法评价艾叶油微胶囊的缓释性和稳定性。结果:艾叶油微胶囊制备工艺为乳化时间20 min、乳化转速1 500 r·min-1、凝聚温度45℃、凝聚转速300 r·min-1,按此工艺制备得到的艾叶油微胶囊大小均匀、形状规则、不粘连。结论:采用复凝聚法制备艾叶油微胶囊,工艺稳定、产品成型性好,具有良好的缓释性和稳定性。     

两阶段搅拌转速控制策略发酵生产聚唾液酸

目的:优化聚唾液酸发酵过程的搅拌转速.方法:比较不同搅拌转速对大肠杆菌Escherichia coli K235分批发酵生产聚唾液酸过程的影响.结果:根据发酵前、后期菌体细胞比生长速率和聚唾液酸比合成速率达到最大值所需搅拌转速的不同,提出了两阶段搅拌转速控制策略:发酵前期(0～15h)控制搅拌转速500r/min,发酵中后期控制搅拌转速700r/min.结论:两阶段搅拌转速控制策略使聚唾液酸产量达到3 966mg/L,比恒定搅拌转速500r/min和700r/min分别提高了31.8%和49.3%.将两阶段搅拌转速控制策略与分批补料发酵技术结合,聚唾液酸产量提高到5 108mg/L,山梨醇的转化率达到0.12g/g.     

改善蛋白质药物PELA控释微球释放性能的研究

开展了以乙酸乙酯(EA)与二氯甲烷(MC)的混合溶液为有机溶剂、以单甲氧基聚乙二醇-聚-DL-乳酸(PELA)为膜材的W／O／W复乳-分步固化法制备蛋白质药物控释微球的研究。为解决微球突释率高、且突释后的释药速度缓慢的问题,实现后期快速释放,以溶菌酶为模型蛋白,重点考察了膜材组成、内水相体积以及外水相盐浓度对微球释药速率的影响。结果表明,当外水相盐浓度增大至1.5％时可将释放率由22％提升至45％,是一种较好的加快微球释药速率的途径,因此可通过选择适当的外水相盐浓度,达到所期望的药物释药速率。     

正交法优化保加利亚乳杆菌NQ2508微囊制备工艺

目的制备保加利亚乳杆菌NQ2508双层微胶囊,并考察其包埋产率和耐胃酸能力。方法以保加利亚乳杆菌NQ2508改性淀粉微胶囊为研究对象,以聚丙烯酸树脂为包衣材料,采用流化床底喷工艺制备微胶囊,通过正交试验考察进风温度、雾化压力、进风风量、包衣增重4个因素对微胶囊化的影响。结果最佳工艺条件为进风温度50℃、雾化压力2.0bar、进风风量35m3/h、包衣增重30%,在该工艺条件下制得的微胶囊包埋产率为50.2%,微胶囊经人工胃酸处理后活菌存活率为39.2%。结论工艺优化后制得保加利亚乳杆菌NQ2508双层微胶囊的包埋产率和耐酸能力均较高。     

生物破乳菌的筛选及发酵条件的优化

生物破乳剂的开发可以降低油田乳状液对石油工业和生态环境的负面影响并减少化学破乳剂的使用量。本研究建立了一套高效、便捷的破乳菌筛选方法, 并对破乳菌的特性进行研究。利用大庆油田受石油污染土壤作为菌种来源, 将Tween 60-water(0.072%, V/V)和Span 60-oil (0.028%, V/V)以6.5:3.5体积比配置出可以稳定200 h以上的O/W型乳状液, 用于破乳菌破乳效能的评价。经过分离纯化、血平板试验、排油试验和破乳试验最终筛选出2株24 h平均排油率在80%以上的优势破乳菌, 初步鉴定为芽孢杆菌属(Bascillus)。通过该破乳菌发酵条的件优化得到, 当温度为25°C, 摇床转数为160 r/min, pH值为9, 接菌量为20%时对该破乳菌生长速率最快, 积累发酵产物的量最多; 当温度为35°C, 摇床转数为120 r/min, pH值为9, 接菌量为2%时该破乳菌代谢产物的破乳活性最高。     

Cr2O3-TiO2吸附产酸克雷伯氏菌HP1氢酶的可见光光解水产氢研究

以钛酸四丁酯和重铬酸铵为原料,采用溶胶凝胶法制备了Cr2O3-TiO2,利用浸渍吸附法将产酸克雷伯氏茵氢酶与Cr2O3-TiO2偶联.研究了搅拌速率、pH、温度等条件对Cr2O3-TiO2吸附氢酶的影响.结果表明,Cr2O2-TiO2在270、440和600nm附近有明显的吸收峰.Cr2O3-TiO2吸附氢酶的最佳条件为温度37℃,pH7.0,搅拌速率100 r/min,该条件下氢酶的吸附率达到80％以上.在氢酶负载量为10％( W/W)、60W白炽灯光源光照度条件下,Cr2O3-TiO2-氢酶催化光解水产氢速率为10 tL/min·g,是Cr2O3-TiO2催化光解水产氢速率(3μL/min.g)的3.33倍,反应体系中加入终浓度为0.05 mmol/L的甲基紫晶(methyl viologen,MV)及0.05mmol/LNa2S2O4可显著提高Cr2O3-TiO2-氢酶光催化产氢速率,达110 μL/min·g.在相同条件下,P25型TiO2仅有微量氢产生.结果表明,Cr2O3-TiO2能利用可见光光解水产氢,氢酶与Cr2O3-TiO2偶联可显著提高可见光光解水产氢活性.     

木聚糖酶发酵工艺在50L罐中的放大

自制的酵母水解液成功替代有机N源酵母浸膏被应用于木聚糖酶发酵,大大降低了原料成本。在此基础上,于50 L罐中进行发酵工艺放大,得到最佳发酵条件:搅拌转速220 r/min、空气流量23 L/min、初始pH 5.5、温度30℃、罐压0.04 MPa,最终发现产酶水平可达到2 864 U/mL,用箭叶圆盘涡轮搅拌桨代替上层平叶圆盘涡轮搅拌桨,产酶水平无显著变化,搅拌功率节约11%。     

Determination of tramadol in human plasma and urine samples using liquid phase microextraction with back extraction combined with high performance liquid chromatography

Liquid phase microextraction by back extraction (LPME-BE) combined with high performance liquid chromatography (HPLC)-fluorescence detection was developed for the determination of tramadol in human plasma. Tramadol was extracted from 2 mL of basic sample solution (donor phase) with pH 11.5 through a micro liter-size organic solvent phase (100 microL n-octane) for 25 min and finally into a 3.5 microL acidic aqueous acceptor microdrop with pH 2.5 suspended in the organic phase from the tip of a HPLC microsyringe needle for 15 min with the stirring rate of 1250 rpm. After extraction for a period of time, the microdrop was taken back into the syringe and injected into HPLC. Effected the experimental parameters such as the nature of the extracting solvent and its volume, sample temperature, stirring rate, volume of the acceptor phase, pH and extraction time on LPME-BE efficiency was investigated. At the optimized condition, enrichment factor of 366 and detection limit (LOD) of 0.12 microg L(-1) were obtained. The calibration curve was linear (r=0.999) in the concentration range of 0.3-130 microg L(-1). Within-day relative standard deviation RSD (S/N=3) and between-day RSD were 3.16% and 6.29%, respectively. The method was successfully applied to determine the concentration of tramadol in the plasma and urine samples and satisfactory results were obtained.     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Effect of alpha-fluoromethylhistidine on the histamine content of the brain of W/Wv mice devoid of mast cells: turnover of brain histamine

In the brains of W/Wv mutant mice that have no mast cells, the histidine decarboxylase (HDC) level is as high as in the brain of congenic normal mice (+/+), but the histamine content is 53% of that of +/+ mice. The effects of alpha-fluoromethylhistidine (alpha-FMH) on the HDC activity and histamine content of the brain of W/Wv and +/+ mice were examined. In both strains, 30 min after i.p. injection of alpha-FMH the HDC activity of the brain had decreased to 10% of that in untreated mice. The histamine content decreased more gradually, and after 6 h about half of the control level remained in +/+ mice, whereas histamine had disappeared almost completely in W/Wv mice. It is concluded that the portion of the histamine content that was depleted by HDC inhibitor in a short time is derived from non-mast cells, probably neural cells. The half-life of histamine in the brain of W/Wv mice was estimated from the time-dependent decrease in the histamine content of the brain after administration of alpha-FMH: 48 min in the forebrain, 103 min in the midbrain, and 66 min in the hindbrain.     

Synthesis of propyl gallate by mycelium-bound tannase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in organic solvent

Aspergillus niger with mycelium-bound tannase activity was employed to investigate the synthesis of propyl gallate from gallic acid and 1-propanol in organic solvents. The effects of various organic solvents (log P: −1.0 to 6.6) on the enzymatic reactions showed that benzene (log P: 2.0) was the most suitable solvent. The water content and protonation state of mycelium-bound enzyme both had significant effects on the activity of tannase. The maximum molar conversion (65%) was achieved with 7.3% (v/v) 1-propanol and 5.56 mM gallic acid at stirring speeds of 200 rev/min, 40 °C in presence of anhydrous sodium sulfate and PEG-10,000. Enzyme specificity for the alcohol portion (C₁–C₈) of the ester showed that the optimum synthesis was observed with alcohols ranging from C₃ to C₅.     

Histochemistry of 3beta-hydroxysteroid dehydrogenase in rat ovary. I. Amethodological study

By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3beta-HSD was investigated: 1. Prefixation of small tissue blocks with 1% W/V methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced. 2. Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% W/V for 5 min) thereby enhancing the Nothing dehydrogenase reaction. 3. Preextraction of lipids with acetone for 20 min at -30 degree C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells). 4. No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20 degree C. 5. By using the double-section incubation method no diffusion of 3beta-HSD or rediffusion of NADH or PMSH could be noticed withn 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0mg/ml). 6. The utlity of different inhibitors of alkaline phosphomonoesterase was tested and discussed. 7. By inhibiting alkaline phosphomonoesterase with 0.1 mM of L-p-bromotetramisole or 16 mM of beta-glycerophosphate, 3beta-HSD was shown to be exclusively NAD-linked. 8. Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3 beta-HSD, but not of NADPH-tetrazolium reductase. 9. 3beta-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited by N-ethyl maleimide. 10. Whether alcohol dehydrogenases may use steroids as substrate is discussed; It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed. 11. Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase. 12. Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF). 13. The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone. 14. If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.     

Molecular Dynamics Simulations and Principal Component Analysis on Human Laforin Mutation W32G and W32G/K87A

Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure–function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.