不同酸水解酪蛋白对W135群和Y群脑膜炎奈瑟菌荚膜多糖产量的影响

目的 探索不同酸水解酪蛋白对W135群与Y群脑膜炎奈瑟菌(脑膜炎球菌)荚膜多糖产量的影响。方法 分别以NaCl质量分数为37%和14%的酸水解酪蛋白作为有机氮源配制改良半综合高盐培养基和低盐培养基,利用全自动细菌发酵罐分别在两种培养基里培养W135群和Y群脑膜炎球菌,比较这两种菌株在两种培养基中的生长时间、收获液菌密度( A 600 nm 值)、收获液去菌体后与去复合多糖后上清中的荚膜多糖含量,以及纯化后的精糖产量;比较高盐培养基收获液及其2倍稀释液中不同终含量的十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)溶液对多糖沉淀效果的影响。结果 W135群与Y群脑膜炎球菌在两种培养基中的培养时间差异无统计学意义( P >0.05),但高盐培养基收获液的菌密度、收获液去菌体后上清液中的多糖含量均高于低盐培养基收获液,差异有统计学意义( P < 0.05),而高盐培养基收获液纯化后的精糖产量低于低盐培养基,差异有统计学意义( P <0.05)。高盐培养液2倍稀释后,在CTAB终体积分数为0.04%时,W135群与Y群脑膜炎球菌多糖全部沉淀,而未稀释高盐培养液即使CTAB终体积分数提高到0.14%,依然也不能完全沉淀 W135群与Y群脑膜炎球菌多糖。结论 不同酸水解酪蛋白对W135群和Y群脑膜炎球菌的生长密度和荚膜多糖产量有影响,用CTAB溶液沉淀荚膜多糖时需控制收获液盐含量。     

脑膜炎球菌多糖疫苗培养基的优化研究

目的探索A群、C群脑膜炎球菌多糖疫苗培养基的适宜配方。方法通过筛选改良培养基(配方2)中酸水解酪蛋白替代培养基配方1中原50%盐酸酪蛋白水解液制备相应的培养基,培养A群、C群脑膜炎球菌一定时间后,以收获的细菌浓度和复合多糖量来确定培养基的配比,并比较该培养基在不同温度条件下培养细菌的结果。结果在A群、C群脑膜炎球菌多糖疫苗不同培养基的细菌培养过程中,用酸水解酪蛋白制备的改良培养基(配方2)培养的细菌浓度和多糖收获量均高于其他培养基(配方1和配方3),用酸水解酪蛋白培养基能提高脑膜炎球菌的产量。结论以酸水解酪蛋白为主要原料(配方2)的改良培养基能作为流脑A群、C群细菌的最适培养基,且细菌在(37±0.2)℃培养情况良好。     

离子色谱法测定ACYW135群脑膜炎球菌结合物中Y群及W135群多糖含量

目的利用离子色谱法即高效阴离子交换柱层析—脉冲安培检测法(HPAEC-PAD),测定ACYW135脑膜炎球菌多糖蛋白结合物中Y群和W135群多糖含量的方法,并加以验证。方法将ACYW135脑膜炎球菌多糖蛋白结合物中Y群和W135群多糖用三氟乙酸(TFA)水解为特异性单糖(葡萄糖、半乳糖),并去除水解液中残留的TFA,用HPAEC-PAD分析检测,用PA10糖分析柱分离单糖,电化学检测器测定葡萄糖及半乳糖含量,用Chromeleon色谱工作站记录并分析数据。对上述方法进行专属性、准确性、重复性验证,确定该方法的检出限和定量限。结果 Y群和W135群多糖在TFA2.5 mol/L(终浓度)、90℃、4 h可完全水解为葡萄糖、半乳糖。对照品葡萄糖及半乳糖在0.10~60.00μg/m L范围内,质量浓度和色谱峰面积呈较好的线性关系,r均大于0.99,回收率为87.94%~109.80%,相对标准偏差(RSD)为1.00%~2.00%,检出限为0.05μg/m L(信噪比3∶1),定量限为0.10μg/m L(信噪比10∶1)。结论离子色谱法可同时检测脑膜炎球菌多糖蛋白结合物中Y群和W135群糖含量,该方法操作简便、灵敏、快速,干扰小,重现性好,适用于对相关疫苗生产过程中的质量控制。     

2～59岁健康人群接种ACYW135群脑膜炎球菌多糖疫苗的免疫原性观察

目的评价ACYW135群脑膜炎球菌多糖疫苗在2～59岁健康人群中的免疫原性。方法 2～59岁健康人群接种者随机抽样(n=60),接种一剂四价脑膜炎球菌多糖疫苗。采集接种前和接种后1个月血清,采用体外杀菌试验(Serum bactericidal assay,SBA)检测血清中抗A、C、Y、W135群脑膜炎球菌的血清杀菌滴度。结果免疫前、后血清抗A群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为1241(736,2091)和7559(5520,10351)(P<0.05);抗C群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为4(9,21)和4787(2947,7775)(P<0.05);抗W135群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为16(9,28)和368(162,883)(P<0.05);抗Y群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为120(58,246)和1373(687,2745)(P<0.05)。免疫前和免疫后血清抗A群脑膜炎球菌的杀菌滴度≥128的比例分别为87(77.4,95.1)%和100(83.2,100)%;抗C群脑膜炎球菌的比例分别为17(8.3,28.5)%和97(88.5,99.6)%;抗W135群脑膜炎球菌的比例分别为13(5.9,24.6)%和68(55.0,79.7)%;抗Y群脑膜炎球菌的比例分别为57(43.2,69.4)%和85(73.4,92.9)%。免疫后较免疫前抗A群、C群、W135群和Y群脑膜炎球菌杀菌抗体滴度≥4倍升高的比例分别为50(27.2,72.8)%、97(88.5,99.6)%、62(43.2,73.9)%和55(41.6,67.9)%。结论虽然免疫前人群由于地方和国家免疫计划的实施已具有较高水平的抗A群脑膜炎球菌的血清杀菌滴度,但接种ACYW135群脑膜炎球菌多糖疫苗后可以使其保护水平进一步提高,并使人群对C群、W135群和Y群脑膜炎球菌的低水平杀菌抗体滴度均显著升高达到保护水平,证明ACYW135群脑膜炎球菌多糖疫苗在2～59岁健康人群中具有比较好的免疫原性。     

分光光度法标定酵母超滤液浓度及其在脑膜炎球菌培养基优化过程中的应用

目的:建立检测酵母超滤液浓度的方法,并探索适合A、C群脑膜炎球菌生长的无动物源性培养基中酵母超滤液的浓度。方法:通过全波长扫描及不同波长吸光度值的比较确定检测酵母提取物浓度的最佳波长;用3000 Da超滤膜超滤酵母提取物溶液,制备酵母超滤液并检测其性质;观察脑膜炎球菌在含不同批次酵母超滤液的半综合培养基中的生长情况,验证不同批次标化的酵母超滤液的稳定性;配制含不同浓度酵母超滤液的半综合液体培养基,观察A、C群脑膜炎球菌的生长情况。结果:通过全波长扫描及不同波长吸光度值的比较,确定了检测酵母提取物浓度的最佳波长为405 nm;采用3000 Da超滤膜超滤后的酵母超滤液不与CTAB产生沉淀,通过检测其D_(405nm)值,可以确定酵母超滤液的浓度;从生长情况考虑,A、C群脑膜炎球菌半综合培养基中最优酵母超滤液浓度分别为4、2 mg/L。结论:建立了制备酵母超滤液及分光光度法标定酵母超滤液浓度的方法,确定了培养A、C群脑膜炎球菌半综合培养基中最优酵母超滤液浓度。     

尖顶羊肚菌液体培养基质与条件的研究

通过对尖顶羊肚菌液体培养基质与条件的研究,明确其菌丝生长的最适pH值、最适温度、适宜光照条件、适宜葡萄糖和蛋白胨浓度、适宜培养基,以便应用于尖顶羊肚菌液体菌种的生产和工业发酵。结果表明:菌丝的最适生长温度为2 5℃;最适生长pH值为6 ;葡萄糖和蛋白胨最适浓度分别为2 0 0g/L和10g/L ;菌丝在黑暗环境下生长良好,光照对菌丝生长具有抑制作用;用胡萝卜酵母膏培养基振荡培养形成的菌丝球多,菌丝生长量大;菌丝球在不同培养基中生长,可引起培养液pH值的上升或者下降;菌丝球可利用培养基内的氨基酸,使氨基酸降解,在胡萝卜酵母膏培养基中振荡培养8d的菌液总氨基酸含量较原液减少了36 71% ,亮氨酸、异亮氨酸和甲硫氨酸含量的下降幅度最大     

B群链球菌培养条件的优化研究

利用B群链球菌(Streptococcus Group B,SGB)有可能发展一种新的预防用疫苗。为研究其生长特性,对该菌在不同培养条件下的生长状况进行了研究。分别考察了液体培养基、接种量、pH值、生长因子、溶氧等因素对SGB生长的影响。结果发现：SGB液体培养基生长因子中尿嘧啶、烟酸、泛酸钙等对SGB的生长有较大影响,葡萄糖最佳浓度为14g/L;此外,发酵培养最佳接种量为0．5～0．8亿／ml;最适pH值为7～8,培养过程中调节pH、补加葡萄糖能维持SGB继续生长;增大溶氧对SGB生长影响不明显。在此优化基础上,连续进行3批100L发酵,SGB菌生长良好,荚膜多糖产量可观。     

溶氧浓度对A群脑膜炎球菌荚膜多糖产量及质量的影响

目的研究不同溶氧(dissolved oxygen, DO)浓度对A群脑膜炎球菌(Meningococci group A)荚膜多糖产量及质量的影响,确定A群脑膜炎球菌发酵时的最适DO浓度。方法通过调整发酵罐搅拌转速,通气量和罐内压力,使得A群脑膜炎球菌发酵时发酵液中的DO浓度分别为5%、10%、20%和30%,测定不同DO浓度细菌的生长情况、葡萄糖和氢氧化钠消耗情况,发酵结束后从发酵液中提纯荚膜多糖,比较不同DO浓度荚膜多糖的产量和质量。结果 DO浓度对A群脑膜炎球菌的生长及代谢影响显著,当DO浓度为20%时,发酵液中细菌的终浓度是DO浓度5%的1.9倍,是DO浓度10%的1.3倍;发酵液中的葡萄糖消耗速率和氢氧化钠的用量均随着DO浓度的升高而降低。DO浓度对A群脑膜炎球菌荚膜多糖的产量及质量也有明显影响。当DO浓度为20%时,A群脑膜炎球菌荚膜多糖产量是DO浓度为5%的3倍,是DO浓度为10%的1.7倍;荚膜多糖细菌内毒素含量较DO浓度为5%下降78%,较DO浓度为10%下降64%;分子大小也优于其他溶氧浓度。结论 DO浓度为20%是A群脑膜炎球菌发酵时的最适DO浓度。     

ACYW135群脑膜炎球菌多糖疫苗的免疫原性评价

目的评价ACYW135群脑膜炎球菌多糖疫苗在昆明健康人群接种的免疫原性,为流脑防治策略提供依据。方法 2010年对昆明市2岁!3、岁!、4岁!、5岁!、6岁!、10岁!、≥15岁共7个年龄组分层随机抽取筛选出654名健康人,分别采集免前和免后1个月血清。用微量杀菌力试验(TTC法)分别检测血清中抗A、C、Y和W135群脑膜炎球菌杀菌抗体的水平。结果免后1个月抗A、C、Y和W135群脑膜炎球菌的杀菌抗体阳转率分别为96.99%、96.37%、88.43%和87.07%,抗A、C、Y和W135群膜炎球菌血清的杀菌抗体几何平均滴度(GMT)分别为1∶297.991、∶195.80、1∶72.74和1∶45.95。结论 ACYW135群脑膜炎球菌多糖疫苗在≥2岁以上的健康人群中有较好的免疫原性。     

不同营养基质与条件对灰树花生长的影响

探讨了不同营养基质与条件对灰树花[Grifola frondosa(Fr.)S.F.Gray]生长的影响。试验结果表明：菌丝生长的最适pH为5-6;最适氮源为蛋白胨、牛肉膏和谷氨酰胺,葡萄糖和蛋白胨最适浓度分别为100和1g/L;菌丝在黑暗环境下生长良好;使用马铃薯蛋白胨培养基进行振荡培养,菌丝球生长较好;母种采用马铃薯麦麸培养基,栽培种选用棉子壳麦麸石培养料,可缩短制种周期,获得质量较高的菌种;菌丝含有较多的钙、铁和锌等元素。     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Effect of interstage mixing in multistage culture systems on continuous biomass production

The significance of the interstage mixing on important process parameters of biomass production was studied. The experiments were performed in a multistage tower fermentor and in fermentors in series. The interstage mixing effect can be evaluated under conditions of geometrical similarity, identity of oxygen transfer rate, and identity of dilution rate per stage in the individual stages of both culture systems. Candida utilis was cultivated on a synthetic medium with ethanol as the sole carbon and energy source in the concentration range 10–100 g/liter. Dilution rate, temperature, and pH in each stage of both culture systems were kept constant. It was demonstrated that in the multistage tower fermentor the definite backflow which ensures the permanent reinoculation by adapted cells significantly decreases the inhibitory effect of higher ethanol concentrations on the cell growth and on the rate of ethanol utilization.     

Effect of fermentation conditions on toxin production by Clostridium botulinum type B.

To obtain high yields of toxin for the preparation of purified neurotoxoids, we examined the time of appearance and the quantity of toxin produced by the Bean strain of Clostridium botulinum type B under various conditions by using a fermentor system. The medium employed consisted of 2.0% casein hydrolylsate and 1.5% yeast extract plus an appropriate concentration of glucose. The maximum toxin concentration (4 x 10(5) to 5 x 10(5) mouse median lethal doses per ml) was attained within 48 h under the following fermentation conditions: an initial glucose concentration of 0.5 or 1.0%, a temperature of 35 degrees C, a nitrogen overlay at a rate of 5 liters/min, and an agitation rate of 50 rpm.     

An evaluation of suspension culture systems for the growth of murine lymphoblastoid lines expressing TL and Thy-1 alloantigens

As a prelude to our studies on TL and Thy-1 differentiation alloantigens, three murine lymphobhastoid cell lines were examined for expression of these components. Optimal conditions for their mass culture were also determined. Several suspension culture systems were evaluated: (a) 50 ml through 500 ml Wheaton and Bellco spinner flasks as well as 1, 4, and 8 liter Wheaton flasks modified for semicontinuous culture conditions, (b) 3 liter Chemapec Vibrofermentor, and (c) 14 liter New Brunswick fermentor. Utilizing these types of vessels the optimal culture conditions were evaluated as to the effect of: (1) pH, (2) initial concentration of cell inoculum, (3) types of media, and (4) methods of gassing and gas mixtures on the rate of growth and alloantigen expression. This study demonstrated that cells could be cultured on a semicontinuous basis up to densities of 2–4 × 10⁶ cells/ml if a vessel of appropriate dimensions was utilized, the appropriate medium selected, and the pH controlled by CO₂ and air overlay. Once these parameters were established the growth of a given cell line was highly reproducible: Under optimal culture conditions the expression of Thy-1 was maximum while the cells were in the exponential stage of growth and reduced during the lag and stationary phases of growth. The expression of TL did not vary as significantly during the various stages of growth. One cell line grown in medium supplemented with 10% horse serum expressed lass Thy-1 than those grown in medium containing 10% fetal calf serum. The factors affecting cell growth and alloantigen expression have been considered in the design of a large-scale suspension culture facility for culturing 1000 liters of cells per week.     

Effect of pH on the batch fermentation of pullulan from sucrose medium

Two strains of the yeast-like fungus Aureobasidium pullulans 2552 and 140B have been used for the fermentative production of the polysaccharide pullulan from a sucrose synthetic medium. In the batch fermentation, either in Erlenmeyers or in the fermentor, the pH of the culture medium was decreased rapidly from its initial pH value of 5.5 to the self-stabilized final value of 2.5 within 24 h. Experiments on the effect of initial pH on the fermentation revealed that at very low initial pH values, such as at pH 2, the polysaccharide production was in-significant. However, the biomass concentration obtained was very high at this very low initial pH value. This interesting phenomenon was served as the basic principle for the development of the bistaged pH fermentation process for the production of pullulan. In this process the first stage of fermentation was conducted at the very acidic pH for the best production of biomass. When the biomass concentration reached its maximum value, the second stage of fermentation was initiated by adjusting the medium pH to a higher value for promoting the synthesis of the polysaccharide. Experiments conducted in Erlenmeyers and in the fermentor confirmed this concept. The bistaged pH process enhanced the polysaccharide concentration in the medium, influenced the rheological properties of the fermentation broth, and has a potential of operation under nonsterile and nonaseptic conditions.     

Growth kinetics and cellulase biosynthesis in the continuous culture of Trichoderma viride.

Continuous culture studies have been carried out growing Trichoderma viride QM 9123 in a 10 liter stirred fermentor on a medium containing commercial glucose as the carbon source. Experiments were carried out at 30 degrees C and at three controlled pH values of 2.5, 3.0, and 4.0 over a range of dilution rates from 0.01 to 0.11 hr-1. Steady-state values of cell, glucose, and cellulase concentration oxygen tension, and outlet gas oxygen partial pressure were recorded. Values of maximum specific growth rate, endogenous metabolism coefficient, Michaelis-Menten coefficient, yield and maintenance coefficient for glucose were derived and correlated the effect of the hydrogen ion concentration. Specific oxygen uptake rates were correlated with specific growth rates and absorption coefficients were shown to be a function of dilution rate independent of pH. Some data on cellulase biosynthesis were examined and correlated in terms of a maturation time model.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Physiological characterictics and energy balance ofKlebsiella aerogenes in a multistage tower fermentor

Biomass growth, consumption of carbon and energy source, specific rates of formation of metabolic byproducts, biomass yield referred to the C-source and to oxygen, respiration rate and the value of RQ were studied in Klebsiella aerogenes CCM 2318 (on a synthetic glucose medium) at different specific growth rates. Maintenance coefficients and the total energy balance of the cultivation process were evaluated for a multistage tower fermentor with a defined interstage mixing. The results pointed to changes in both glucose metabolism and the physiological state of the population, brought about by changes in specific growth rate. As compared with a chemostat, the culture was found to exhibit a different physiological character is stages 1 and 4 despite a considerable interstage mixing.