正交试验优化鼠疫活疫苗冻干稳定剂

目的优化皮上划痕用鼠疫活疫苗的稳定剂配方。方法采用正交试验,以鼠疫杆菌冻干存活率为检测指标,分别对乳糖、谷氨酸钠、硫脲和尿素4种成分组成的稳定剂配方,以及乳糖、蔗糖、谷氨酸钠、硫脲和尿素5种成分组成的稳定剂配方进行优化。结果 4种成分组成的稳定剂最佳组合为乳糖7.5%、谷氨酸钠0.5%、硫脲1.0%、尿素0.1%(均为质量分数),此配方可使菌体的冻干存活率达到(68.49±6.19)%;5种成分组成的冻干稳定剂最佳组合为乳糖7.5%、蔗糖7.5%、谷氨酸钠0.5%、硫脲0.5%、尿素0.1%(均为质量分数),此配方可使菌体的冻干存活率达到(74.71±6.34)%,均高于现有稳定剂配方。结论优化的两种稳定剂均对鼠疫杆菌具有较好的保护作用。     

冻干人用狂犬病疫苗(Vero细胞)稳定剂配方的筛选

目的筛选出适合冻干人用狂犬病疫苗(Vero细胞)的热稳定性好、无明胶的稳定剂配方。方法以水分、外观、效价、热稳定性效价为指标,尤其是效价和热稳定性效价,对A、B、C、D、E、F、G等6种稳定剂配方进行系统的优化组合筛选,6种稳定剂配方分别含有蔗糖、乳糖、人血白蛋白、甘氨酸、精氨酸、明胶、尿素、甘露醇、右旋糖苷,其中A为现行疫苗生产配方。运用单因素五元设计法,筛选出最优稳定剂组合。结果方差分析结果显示D组配方对冻干人用狂犬病疫苗(Vero细胞)的M-ABT结果 13.95,37℃放置4周M-ABT结果 13.05;NIH效价1.94,37℃放置4周NIH效价1.56。结论 D组配方对冻干人用狂犬病疫苗(Vero细胞)有较好的保护作用。     

构巢曲霉冻干菌粉的制备及优化

【背景】目前,用以降解园林绿化废弃物中木质素的菌剂多为液体菌剂或固体菌剂,鲜有对粉状菌剂的研究。【目的】研制高活性冻干菌粉,提高其冻干存活率并优化其工艺,以解决液体菌剂或固体菌剂在运输、储藏及使用上存在的问题。【方法】以一株木质素降解菌构巢曲霉(Aspergillus nidulans)为研究对象,利用真空冷冻干燥法制备冻干菌粉。以菌株的冻干存活率为评价指标,通过单因素试验筛选适于菌株冻干过程的保护剂种类及浓度梯度,再通过正交试验优化冻干菌粉复合保护剂配方。获得配方后,进一步探究冻干菌粉的复水条件和储藏条件。【结果】保护效果较优的4种保护剂成分经复配后对冻干存活率的影响顺序为蔗糖>葡萄糖>脱脂乳粉> α-乳糖。经优化后的保护剂配方以蔗糖15%、葡萄糖1%、α-乳糖10%、脱脂乳粉1%为最佳;复水条件以生理盐水为溶剂,复水30 min为最优。在此条件下制备和使用冻干菌粉,菌株的冻干存活率可达83.33%,有效活菌数可达1.2×10¹⁰ CFU/g。最佳储藏温度为-20 ℃,在此温度下保存28 d后,菌粉活性无明显下降。【结论】该研究获得的制备和储藏构巢曲霉冻干菌粉条件,具有菌株损失率低、可长时间保存的特点,对推进木质素降解菌在实际生产中应用具有积极作用。     

冻干保护剂对假单胞菌菌粉存活率的影响

保护剂的添加对提高假单胞菌冷冻干燥菌粉的存活率和贮藏稳定性有显著效果。设计实验对冻干保护剂进行筛选和优化研究,通过单因素实验和正交实验筛选出了最佳保护剂组合:脱脂乳粉10%,山梨醇3%,甘油1%,L-抗坏血酸钠2%,假单胞菌冻干后的存活率可达90%,菌粉25℃存放6个月,存活率为50.2%。     

冻干高活力纳豆芽胞杆菌菌粉保护剂的筛选和优化

【目的】对冻干高活力纳豆芽胞杆菌菌粉保护剂进行筛选和优化研究,提高菌粉活菌存活率。【方法】采用单因素实验和正交实验设计,通过测定活菌存活率,筛选出最佳保护剂的配方;并研究采用优化后冻干保护剂制备的菌粉在20°C、4°C、25°C下的保存稳定性。【结果】纳豆芽胞杆菌的有效保护剂是:脱脂乳粉、甘露醇、L-抗坏血酸钠。最佳冷冻干燥保护剂配方是:脱脂乳粉10%+甘露醇4%+L-抗坏血酸钠1%,存活率达到91.63%。菌粉在20°C、4°C、25°C下保存12个月后,存活率分别为:88.79%、70.16%和10.52%,说明菌粉在20°C和4°C下保存稳定性较好,25°C下稳定性比较差。【结论】对纳豆芽胞杆菌冻干菌粉保护剂的优化,对纳豆芽胞杆菌的应用、活菌产品的质量稳定及新产品的研发均有一定的指导意义。     

降胆固醇益生菌发酵剂的制备

以前期筛选出来的降胆固醇益生菌嗜酸乳杆菌S-59为出发菌株，对冻干发酵剂的制备工艺进行研究和优化。结果表明：菌悬液的离心收集条件为5 000 r/min、20 min；通过单因素试验和响应面优化试验，确定冻干保护剂为脱脂乳8.95%、海藻糖5.24%、甘油4.54%，细胞存活率可达90.1%。冻干发酵剂的保藏条件为在真空条件下-4℃保藏。     

裂殖壶菌Schizochytrium sp. 20888产DHA培养条件优化

【目的】为了优化裂殖壶菌产DHA的培养条件,提高油脂中DHA含量。【方法】采用单因素试验和正交设计试验方案,针对分批培养时间、培养基碳、氮源的种类和浓度以及培养温度开展试验,采用重量法测定生物量、采用索氏提取法测定油脂总量,采用气相色谱法测定油脂DHA含量,考察培养条件对细胞油脂DHA含量的影响。【结果】最适培养时间为4 d,培养温度23°C,最优碳氮源组成为(g/L):葡萄糖65、甘油80、蛋白胨6、酵母粉4和谷氨酸钠8。【结论】裂殖壶菌Schizochytrium sp.20888在葡萄糖和甘油组成的复合碳源和由蛋白胨、酵母粉和谷氨酸钠组成的复合氮源的培养基中可以得到最优的DHA产量,细胞DHA含量能达到33.68%。     

芽胞杆菌B13液体发酵培养基的优化

目的建立培养技术,开发芽胞杆菌B13的微生态制剂。方法单因素实验后综合考虑,选择红糖、麸皮、豆粕粉、尿素等4个因素,每个因素3水平进行L9(34)正交试验。结果获得芽胞杆菌B13的最优碳源为葡萄糖,最优氮源为豆粕粉,最佳培养基成分为:红糖1%,麸皮1%,豆粕粉0.5%,尿素1%,磷酸氢二钾0.5%,水1 000 mL,pH 7.0~7.2。结论对最优培养基配方进行有效活菌数计数,芽胞杆菌有效活菌数为7.8×109CFU/mL。     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

野生毛尖蘑的生物学特性及驯化栽培

为了研究固体培养不同条件对野生毛尖蘑菌丝生长的影响,对温度、初始p H、碳源和氮源4个因素进行单因素试验,从中选出3个最优的水平进行正交试验。结果表明,野生毛尖蘑的最适生长温度为16℃,最适初始p H为6.0,最适碳源为蔗糖,最适氮源为酵母浸粉。对毛尖蘑栽培料的配方进行了筛选,试验结果表明:配方为杂木屑30%,玉米芯35%,麦麸25%,豆粕4%,玉米粉3%,石灰1.5%,石膏1.5%的栽培袋,经覆土栽培法可形成子实体。     

Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.     

Identification of immunoreactive proteins of Brucella melitensis by immunoproteomics

Infection with Brucella causes brucellosis, a chronic disease in humans, which induces abortion and sterility in livestock. Among the different Brucella species, Brucella melitensis is considered the most virulent and is the predominant species associated with outbreaks in China. To date, no safe human vaccine is available against Brucella infection. The currently used live vaccines against Brucella in livestock induce antibodies that interfere with the diagnosis of field infection in vaccinated animals, which is harmful to eradication programs. However, there is as yet no complete profile of immunogenic proteins of B. melitensis. Towards the development of a safer, equally efficacious, and field infection-distinguishable vaccine, we used immunoproteomics to identify novel candidate immunogenic proteins from B. melitensis M5. Eighty-eight immunoreactive protein spots from B. melitensis M5 were identified by Western blotting and were assigned to sixty-one proteins by mass spectrometry, including many new immunoreactive proteins such as elongation factor G, F0F1 ATP synthase subunit beta, and OMP1. These provide many candidate immunoreactive proteins for vaccine development.     

Cryopreservation studies of Campylobacter

Seven strains of Campylobacter fetus ss. fetus, one of Campylobacter fetus ss. venerealis, and one of Campylobacter jejuni were preserved using a variety of cryopreservation methods. Organisms were frozen to -150 degrees C in a liquid nitrogen refrigerator, in the freezer compartment of a refrigerator (-20 degrees C), and in a mechanical freezer (-65 degrees C). In the latter two cases, viabilities of the organisms were compared after being frozen in Brucella Albimi broth and 10% glycerol. Viabilities were also examined after Campylobacter species were freeze-dried using rapid or slow cooling, using sucrose or skim milk as cryoprotective agents and in bulb-type vials on a manifold or batch vials. Preservation in liquid nitrogen resulted in no loss in viability after 4 years storage. When Campylobacter species were frozen at -20 degrees C, no cells were recovered after 1 month storage in Brucella Albimi broth or seven months in glycerol. A 6.5 log decrease in viability resulted after organisms were frozen at -65 degrees and subsequently stored at the same temperature for 2 years. In this case, glycerol had no protective advantage over Brucella Albimi broth. Postpreservation viability of organisms cooled slowly was two logs higher than those cooled rapidly prior to freeze-drying. When skim milk or sucrose were employed as cryoprotective agents during freeze-drying, equal viabilities resulted. Equivalent viabilities were also demonstrated when the bulb type or "batch" vials were utilized for freeze-drying. No significant differences were observed between the viabilities of the three species when a given cryopreservation method was employed.     

Removal of gelatin from live vaccines and DTaP—an ultimate solution for vaccine-related gelatin allergy

From the early 1990s infants started to receive acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP) before live vaccines such as measles, rubella, and mumps vaccines, which contained gelatin as a stabilizer. Then, an increasing number of cases of anaphylactic/allergic reactions to those live vaccines were reported. Almost all these cases had a previous history of receiving three or four doses of DTaP containing gelatin.Anaphylactic/allergic reactions to live measles vaccine were analyzed using information obtained from the Reporting System, a retrospective study, as well as from the Monitoring System, a prospective study. Dramatic decreases in anaphylactic/allergic reactions to live measles vaccines were observed immediately after each manufacturer marketed gelatin-free or gelatin (hypo-allergic)-containing live measles vaccine, and since the end of 1998 reports on anaphylactic/allergic reactions to live measles vaccine have almost ceased.     

Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats

Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.     

Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying

Cultures of 14 lactic acid bacteria species were freeze-dried in 10 or 20% non-fat skim milk and in distilled water containing one of the following additives: bovine albumin, glycogen, dextran, polyethylene glycol (PEG) 1000, PEG 4000, PEG 6000, glycerol, beta-glycerophosphate, sodium glutamate, asparagine, or cysteine. Each of the potential protective agents tested exhibited marked variations in the protection afforded to different species, none of them was effective for the preservation of viability of thermophilic lactobacilli. However, glycerol provided effective protection for L. leichmannii ATCC 4797 (90% survival), while L. bulgaricus ATCC 11842 reached a viability of 78% with 0.04 M cysteine.     

1-O-methyl-rac-glycerol: A new agent for the cryopreservation of mononuclear cells

1-O-methyl-rac-glycerol (1-O-MG), also known as 3-methoxy-1,2-propanediol is a lipophilic derivative of glycerol, and has been studied as a new cryoprotective agent (CPA) for mononuclear blood cells (MNC), a well-established experimental model in cryopreservation. The chemical modification of the glycerol molecule results in improved cryobiological properties, such as membrane permeability, thus allowing easier handling in the freeze/thaw process. The optimum preincubation period for MNC and 1-O-MG before freezing is 5 min at 4 degree C, resulting in 86% recovery of viable cells, whereas optimal recovery of glycerol-frozen MNC is only guaranteed after 30 min of preincubation at room temperature (74% viable recovery). The optimal concentration of 1-O-MG is 10% (v/v). Although this new agent offers no improvement in cryoprotective properties over dimethyl sulfoxide (Me2SO) there may be possible pharmacological advantages when used in humans. It is, however, obviously superior to glycerol with regard to its permeation kinetics. 1-O-MG might therefore also be of interest in the cryoprotection of other hematopoietic cells and biological tissues.     

Production and control of freeze-dried Brucella abortus strain 19 vaccine by using cells produced by fermentation units

Summary A method of producing freeze-dried Brucella abortus Strain 19 vaccine by using batch cultures on fermentation units is described. Details of the equipment and technique employed, results on microbiological and biological controls are given.