In vitro stability studies of a Factor IX concentrate (Bebulin)

Factor IX: C activity decay was studied in lyophilized concentrates (Bebulin, IMMUNO) stored at room temperature and in reconstituted preparations frozen and stored at -20 degrees C. A very slight, practically identical, decay of Factor IX: C was found in lyophilized concentrates kept at room temperature, in reconstituted lyophilized concentrates frozen and stored at -20 degrees C and lyophilized concentrates kept at +4 degrees C. At the end of the experiment, which was 4-5 months after the expiration date of the product, a 15-30% decay of Factor IX: C could be found in the concentrates with respect to initial levels.     

重组人尿激酶原冻干产品的稳定性

目的：探讨重组人尿激酶原（rhPro-UK）冻干产品的稳定性。方法：采用S-2444发色底物法测定贮存在4℃和-20℃的rhPro-UK冻干产品的活性、单链比例随时间的变化规律;用SDS-PAGE及RP-HPLC肽图分析贮存在-20℃的rhPro-UK冻干产品的结构与组成的变化。结果：4℃保存3年后的rhPro-UK冻干产品的总活性和单链比例基本没有变化,但随着贮存时间的延长,有部分产品降解,如贮存78个月的样品,总活性可降低13％～15％;-20℃保存78个月后,rhPro-UK冻干产品的总活性和单链比例未见明显变化。SDS-PAGE及RP-HPLC肽图图谱显示,-20℃贮存78个月后的rhPro-UK冻干产品的组成和结构没有变化。结论：rhPro-UK冻干产品在4℃的贮存寿命可达3年;长期贮存于-20℃的rhPro-UK冻干产品,其总活性、单链比例及结构组成非常稳定。     

Stable concentrated emulsions of the 1-monoglyceride of capric acid (monocaprin) with microbicidal activities against the food-borne bacteria Campylobacter jejuni, Salmonella spp., and Escherichia coli

Of 11 fatty acids and monoglycerides tested against Campylobacter jejuni, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against C. jejuni upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log(10) reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of Campylobacter isolates from humans and poultry and also killed strains of Campylobacter coli and Campylobacter lari, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log(10) reduction in viable bacterial counts of Salmonella spp. and Escherichia coli in 10 min. C. jejuni was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to Campylobacter-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.     

长双歧杆菌DD98冻干保护剂优化及菌粉保存稳定性研究

以长双歧杆菌DD98为研究对象,通过对冻干保护剂配方的优化,冻干菌粉的存活率提高到90%以上。通过进一步稳定性研究,采用保护剂优化配方制备的冻干菌粉在4℃保存24个月后,活菌数仍在1.0×10^10 CFU/g以上,在25℃条件下可以保存12个月,双歧杆菌的存活率在1.0×10^6CFU/g以上,符合FAO/WHO建议食品益生菌活菌数应在1.0×10^6 CFU/g^1.0×10^7CFU/g的标准。     

Preparation and regeneration of protoplasts from Bacillus megaterium cells dried by sublimation]

Bacterial protoplasts are widely used in genetical research, for instance, in protoplasts fusion experiments and the transfer of heterologous DNA into bacterial cells. The usage of a new fresh grown culture of bacteria in every experiment restricts the reproducibility of the results preventing the technique becoming widespread. The use of antioxidants as components of stabilizing medium for sublimation drying of Bacillus megaterium cells supported cellular viability in bacterial culture. It also made possible preservation of such cellular fundamental properties as the ability to form protoplasts and regenerate the cell wall. Efficiencies of protoplasts formation and generation are similar for lyophilized and fresh grown cells. Cellular properties are conserved for 6 months of storage at least. Experiments with a lot of lyophilized biomass samples are highly reproducible. The potential of the technique was demonstrated in obtaining the hybrid Bacillus megaterium colonies by fusion of protoplasts derived from lyophilized genetically marked strains stored for up to 6 months.     

Survival of Bacteria in Lyophilized Plasma

Although lyophilization reduces the bacterial population in plasma, some species survive for several months. Sensitivity or resistance to lyophilization in plasma is a characteristic of each morphological type, with gram-negative species being more sensitive to destruction than cocci or sporulating gram-positive species. Reconstituted preparations of lyophilized plasma must be incubated for 24 hr to prevent false-negative sterility tests. The killing process in lyophilized plasma continues with time if the preparations are stored at room temperature. Strict asepsis must be realized during processing of plasma because lyophilization alone does not destroy the microorganisms present.     

Lyophilization of rumen fluid for use in culture media

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Development and applications of surface plasmon resonance-based von Willebrand factor-collagen binding assay

Von Willebrand factor (vWf) functions both as a carrier of factor VIII (fVIII) in plasma and as an adhesive protein providing the primary link between collagen of the extracellular matrix and platelets sequestered from blood flow. The functional activity of vWf correlates with the level of its binding to collagen, which is commonly measured in the enzyme-linked immunosorbent assay (ELISA). We developed an automated collagen-binding assay employing the surface plasmon resonance (SPR) phenomenon, which allows one to quantitatively measure the binding of purified vWf and vWf-containing therapeutic fVIII concentrates to collagen type III immobilized on a biosensor chip. The results of the SPR-based assay highly correlated (r = 0.987) with collagen-binding ELISA. The advantages of the SPR-based assay are its higher accuracy and reproducibility in comparison with ELISA. We applied the developed assay for monitoring structural changes in the vWf component of plasma-derived fVIII/vWf concentrates during a virus inactivation procedure performed by heat treatment. We determined the critical residual moisture content of 2% that can be present in lyophilized concentrates during heat-treatment procedures without causing deteriorative changes in vWf properties. Our data suggest that the SPR-based assay is a useful tool in the development of industrial virus-inactivation procedures, allowing one to preserve vWf activity and achieve the maximal therapeutic efficacy of fVIII/vWf concentrates.     

Lyophilization of rumen fluid for use in culture media.

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Stable Concentrated Emulsions of the 1-Monoglyceride of Capric Acid (Monocaprin) with Microbicidal Activities against the Food-Borne Bacteria Campylobacter jejuni, Salmonella spp., and Escherichia coli

Of 11 fatty acids and monoglycerides tested against Campylobacter jejuni, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against C. jejuni upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log₁₀ reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of Campylobacter isolates from humans and poultry and also killed strains of Campylobacter coli and Campylobacter lari, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log₁₀ reduction in viable bacterial counts of Salmonella spp. and Escherichia coli in 10 min. C. jejuni was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to Campylobacter-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.     

冻干神经生长因子活性稳定性试验研究

用不同的物质作保护剂冻干纯化鼠神经生长因子,经检定以人血白蛋白作为赋形剂和保护剂冻干神经生长因子制品外观洁白细腻,结构强度良好,生物活性符合要求,残存水分低于３％。采用留样观察法检测其稳定性,将制品放置４℃～８℃,在０、０．５、１、２、３、６、９、１２、１８、２４、２５个月分别测定其各自的活性。结果表明神经生长因子冻干制品在４℃～８℃保存,２５个月仍然保持原有生物活性     

Extracellular dextranase from Streptococcus oralis.

A human dental plaque organism, Streptococcus oralis (S. mitior), was cultivated in a dextran-free, dialysed medium, and dextranase activity was isolated from the cell-free, culture supernatant. The lyophilized, crude enzyme preparation, optimum pH 6, was subjected sequentially to anion exchange and gel filtration fast protein liquid chromatography (FPLC). The dextranolytic fraction from gel filtration FPLC produced a symmetrical, baseline resolved peak. The dextranolytic enzyme was purified 1,126-fold with a yield of 2.4%. Amino acid analysis revealed a large proportion of alanine and an abundance of acidic amino acids. This extracellular enzyme isolated from S. oralis is constitutive and has a relative molecular mass of 45 kD. Further investigation of the possible structural and biochemical effects of endogenous bacterial glucanases in human dental plaques is necessary.     

来源于E.coli和CHO表达系统的重组人β-NGF性质比较

基于原核表达系统极难表达具有正确三级结构的重组人β神经生长因子（rhNGF-β）,E. coli是否能作为其工业化生产菌株存在争议。为此,将构建于E.coli体系,经表达体外复性后的rhNGF-β与中国仓鼠卵巢细胞（CHO）表达体系分泌的rhNGF-β,运用HPLC、SDS-PAGE、质谱、N-末端分析等手段以及鸡胚背根神经节（DRG）、 PC12 细胞体外测活法对制备产物进行分析、测定。结果表明：二者具有一致的理化性质和生物学活性,HPLC纯度均大于95%、生物学比活均不低于1.8 ng/U;经添加赋型剂制成冻干粉后,在温度37℃±2℃、相对湿度75%±5%条件下进行3个月加速试验,活性均不变。说明E. coli表达体系可生产质量均一、高生物学活性的rhNGF-β,且具有明显成本优势。     

Heat Resistance and Population Stability of Lyophilized Bacillus subtilis Spores

Bacillus subtilis 5230 spores were lyophilized in 0.067 M phosphate buffer and stored at 2 to 8°C for 9 to 27 months. The lyophilized spores were reconstituted with buffer or 0.9% saline, and the heat resistance was determined in a thermoresistometer. Lyophilization had no effect on the heat resistance of the spores but did result in a slight decrease in population (≤0.3-logarithm reduction). The lyophilized spores maintained heat resistance and population levels over the test periods. The D-values ranged from 0.44 to 0.54 min at 121.1°C, and the z-values ranged from 6.1 to 6.6°C. Lyophilization was concluded to be an acceptable alternative for storage of bacterial spores that are to be used as biological indicators in sterilization processes.     

Mass spectral analysis of complex lipids desorbed directly from lyophilized membranes and cells

Three desorption ionization techniques--laser desorption, plasma desorption and fast atom bombardment mass spectrometry--have been applied to lyophilized cells, membranes, lysed cells and various extracts. It has been shown that intact polar lipids are selectively desorbed from biological membranes by these methods and that their mass spectra provide "fingerprints" which reflect the unique biochemical composition of each class of cell or membrane.     

大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。     

Studies on Destabilization of Caseinate Complex during Frozen Storage of Milk

Unpasteurized skim milk was storaged in a frozen state at ?7°C or ?20°C for up to several months. There was no increase of non casein and non protein nitrogens, but a slight increase of free tyrosine and a slight decrease of alkaline phosphatase activity were detected when storage period was prolonged. Destabilization occurred solely in caseinate complex, but non micellar casein appeared to be stable.

The contents of calcium and inorganic phosphate in the caseinate complex separated by ultracentrifugation were increased appreciably after frozen storage. The viscosity characteristics of frozen storaged skim milk was also investigated.

Caseinate complex was ultracentrifugally separated from skim milk before and after frozen storage, and then lyophilized. Skim milk itself was also lyophilized before and after frozen storage. Dispersibility was examined on the reconstituted suspension of the lyophilized samples.

The lyophilized sample from frozen storaged milk was much less dispersible than the lyophilized control sample prepared before frozen storage. However, when lyophilized samples were once resolved with reagents such as urea and potassium oxalate and then dialyzed against fresh milk, stable micelle resulted in both samples prepared before and after frozen storage.

Some reduction of dispersibility occurred during lyophilization and subsequent storage in a dried state in the caseinate complex prepared before frozen storage. This reduction was small when skim milk was lyophilized and stored.     

人心型脂肪酸结合蛋白(H-FABP)的原核表达、纯化和冻干品的制备

目的：获得重组人心型脂肪酸结合蛋白,分析其活性及制备冻干品。方法：从GenBank中检索人源H-FABP CDs 序列,合成基因后构建原核表达载体pET-28a（+）-H-FABP。将表达载体转入E.coli BL21（DE3）,摸索pET-28a（+）-H-FABP最佳诱导条件,表达并纯化重组蛋白。使用检测试剂检测纯化后的重组H-FABP活性,研究最佳冻干方案并考察冻干品的稳定性。结果：经过优化诱导条件,重组H-FABP在BL21（DE3）中以可溶性蛋白形式表达。诱导条件为OD₆₀₀≈0.6,IPTG终浓度0.4mmol/L,30℃诱导4h。通过Ni²⁺亲和层析纯化可得到纯度大于95%的重组H-FABP蛋白。重组H-FABP冻干品可以在37℃稳定保存12d,25℃、4℃稳定保存至少4个月。结论：本项研究中的重组H-FABP表达体系成熟、蛋白活性高,冻干品稳定性好,为后续研究提供了稳定高效的生物原材料。     

Preservation of Functional Mammalian Polysomes by Lyophilization

LYOPHILIZATION has been used to preserve viable microorganisms for extended periods of time¹. Recently, it has been reported that ribosomes functional in in vitro protein synthesis can be isolated from lyophilized fungi² and that lyophilized Escherichia coliribosomes retain fully their capacity for poly U-directed phenylalanine incorporation after 5 months storage at room temperature over P₂O₅ (ref. 3). We have now compared the sedimentation profiles and poly U-directed phenylalanine incorporating activity of three types of rabbit reticulocyte ribosome preparations immediately after isolation and after freezing or lyophilization and storage for various times.