吸附无细胞百白破(DPTa)的稳定性试验

采用半综合液体培养基（含０．０５％ＭｅβＣＤ）,大罐培养百日咳Ⅰ相ＣＳ菌制备的吸附无细胞百日咳菌苗、白喉、破伤风类毒素混合制剂（ＤＰＴａ）置４－８℃分别保存一年、保存两年,测三种抗原成分的效力及毒性,以及将该制剂于３７℃分别放置三周、三个月,测定有无毒性逆转。结果表明,该制剂质量稳定,无毒性逆转     

光密度测定法在百日咳杆菌培养菌液浓度测定中的应用

本实验通过抽取百日咳杆菌悬液样品,分别用比浊法和光密度法测量其浓度,并进行各种比较,证明在百日咳杆菌培养中使用分光光度计测量菌悬液光密度值来代替比浊法测定菌悬液浓度的方法是准确可行的,并能建立光密度值与菌悬液浓度的关系,从而提高工作效率、更好的控制培养参数。试验数据表明,利用光密度法测定的百日咳杆菌悬液浓度数据稳定,重复性好,可信度高。证明了光密度法在百日咳杆菌悬液的浓度测定中的可行性,初步建立了光密度值与菌悬液浓度之间的联系。     

適应性百日咳菌种的獲得与应用

1．用多次傅代方法在六号培养基上,经20代傅代所獲得之CS20菌株,虽仍为一相百日咳桿菌,但未獲得在含腺培养基上生是稳定之適应性。 2．C及P5菌株在不適宜的情况下长期培养而獲得之Cv及P5v菌株,经多次全面检查仍为一相百日咳桿菌,合乎生物制品法规之要求。在血清学、毒力、毒性、免疫力等方面与原菌株C及P5均一致,但在含腖之四号培养基上生长稳定有顯著之適应性。 3．雨年来100批大量生产百日咳原液中充分澄明了Cv及P5v在四号培养基上之適应性,且在免疫力方面与原菌株所作原液无差别。因Cv及P5v生是稳定,故较原菌株C及P5所生产之原液之浓度提高200％以上。该適应性地方性菌株是理想而適合於生产百日咳菌苗用的菌种。     

蜡状芽孢杆菌活菌制剂(促菌生)大罐深层培养的研究

本试验就蜡状芽孢杆菌大罐深层培养法进行了初步研究,基本解决了蜡状芽孢杆菌大罐液体深层培养条件下芽孢生成、适宜培养基的选择、培养方法的建立以及菌体的收集方法等技术问题,大罐深层培养生产工艺的建立较固体培养法产量大为增加,污染耗损降至最低,减少劳动强度和繁琐的操作,而且产品质量得到保证和提高.     

一种肠道硫酸盐还原菌的增菌培养基

目的针对已经分离、纯化的肠道硫酸盐还原菌,建立一种能快速、高效地培养菌体的培养基。方法比较营养丰富的GAM肉汤与常用于培养硫酸盐还原菌的选择性培养基Postgate的培养效果,摸索在GAM肉汤中添加不同浓度的硫酸盐对两种肠道硫酸盐还原菌-Desulfovibrio desulfuricans和Desulfovibrio intestina—zis的培养效果。确定效果最佳的改良GAM培养基配方,并测定在该培养基中D．desulful＇icans的生长曲线。结果与Postgate培养基相比,GAM肉汤能在2d内快速培养D．desulfugicans,但培养至6d时细菌数量大幅降低。在GAM肉汤中添加Na2SO4与FeSO4,在实验浓度范围内,均显著地促进硫酸盐还原菌的生长。在此基础上改良GAM肉汤培养基,培养得到的细菌数量较GAM肉汤显著提高。D．desulfuricans的生长曲线显示,2d时细菌生长达到最高峰,数量可达3．5×10^7 CFU／mL;培养6d,细菌数量为7．3×10^6 CFU／mL。结论基于GAM肉汤改良而得到的增菌培养基,能快速、高效地培养肠道硫酸盐还原菌,为后续进一步研究肠道硫酸盐还原菌的生理功能提供了支持。     

一株虫草头孢菌深层培养条件的研究

采用摇瓶培养的方法,以菌丝体收率为指标,通过单因子实验和正交实验确定了虫草头孢菌最佳培养条件和最佳发酵培养基配方。在相同条件下,优化培养基比原培养基的菌丝体收率提高了31.75%。     

用无血培养基和鲍姜培养基临床分离百日咳杆菌的比较

<正> 自从进行百日咳预防接种以来,百日咳的发病率已有减少。但仍有极少的小型暴发。由于百日咳杆菌不易从临床标本中分离出来,所以诊断通常依靠临床资料作出。尽管鲍姜培养基已广泛应用于营养要求高的百日咳杆菌的临床分离。一般来说,含有新鲜血液的培养基的贮藏期限不长。人们认为成功地分离百日咳杆菌最重要的因素是立即划线接种标本。可是,经济上却不利于把鲍姜培养基一直保持在医院里,因为在医院采取标本     

铁-锰氧化鞘细菌的生境条件及分离鉴定

从水库采集不季节和不同水层的水样及水下输水管道堵塞物刊物分析和分离,表明,铁－锰氧化鞘细菌分布在水库不同水层的低浓度有机营养的水流和水下输水管道的堵塞物中。菌在水库中较适的生境条件在夏秋两季,温度为２１￣２５℃、ｐＨ６．１６￣６．８７,总铁含量〉０．４３ｍｇ／Ｌ和微氧环境的中下层及底层。该菌的采用富集培养和限制营养浓度的选择培养基。富集鞘细菌以尿素－葡萄糖－几机盐培养基和Ｓｔｏｋｅｓ培养基为较有效     

泥浸汁对太湖沉积物中的好氧可培养细菌多样性的影响

【目的】研究添加泥浸汁与否对太湖沉积物中可培养细菌的影响。【方法】采用R2A培养基和添加泥浸汁R2A培养基对沉积物中细菌进行分离培养,16S r RNA基因系统发育分析比较种群结构。【结果】培养基中添加泥浸汁,可使可培养细菌的种类数量增加到1.6倍。16S r RNA基因序列分析表明,培养的优势细菌类群存在明显差别。R2A培养基上生长的细菌主要为厚壁菌门(52%)、放线菌门(24%)、变形菌门(20%)和拟杆菌门(4%),其中大部分细菌与芽孢杆菌属、假单胞菌属、节杆菌属等关系密切;而添加泥浸汁的R2A培养基上生长的细菌则主要为变形菌门(40%)、放线菌门(35%)、厚壁菌门(22.5%)和拟杆菌门(2.5%),与鞘脂单胞菌属、芽孢杆菌属、副球菌属、节杆菌属等关系密切。【结论】添加泥浸汁原始营养因子可提高沉积物中可培养细菌的多样性,提高菌种可培养效率。     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

Inhibition of enterotoxin production by, and growth of enteropathogens in a lactic acid-fermenting cereal gruel

Growth and enterotoxin production of three strains of Campylobacter jejuni or Escherichia coli known to produce heat labile enterotoxins were evaluated when added into a lactic acid-fermenting cereal gruel, togwa. A single strain of each of the enteropathogens was simultaneously inoculated with a lactic acid starter culture (sc) to reach a level of about 107 c.f.u./ml and was left to ferment for 48h. Gruels without sc (control gruel), pure cholera toxin in fermenting or control gruel and the test bacteria inoculated into nutrient broth were used as positive toxin controls; gruel without inoculated test bacteria was the toxin-negative control. Viable colonies were counted by spread plating 0.1ml of gruel subsamples collected at intervals during the fermentation period onto different selective media and the heat labile enterotoxin production was evaluated using an assay on Chinese hamster ovary (CHO) cells. In the fermenting gruels, no viable cells of the C. jejuni and E. coli strains were detected after 8 and 24h incubation, respectively, but inocula increased in number or remained at the initial level in the control gruel and in the nutrient broth. After 24h incubation, all supernatants of control gruels with inoculated bacteria showed enterotoxicity to the CHO cells (indicated by elongation of 20–50% of the cells). No toxin activity was observed in the fermenting gruels with or without added bacteria or in control gruel alone. Pure cholera toxin added to control gruel caused the enterotoxigenic effect in 70–100% of the CHO cells, but no activity was detected when it was added to the fermenting gruel. The CHO cells were affected instead by a low pH level but were not elongated. Adjusting the pH of fermented gruels to approximately neutral levels restored the toxin effect when cholera toxin was added but not in the presence of added test bacteria. We conclude that lactic acid fermented cereal gruels with pH 4 have a high potential to inhibit the growth of enteropathogenic bacteria of the genera C. jejuni and E. coli and to inhibit production of heat labile enterotoxins. Regular consumption of fermented cereal weaning foods will therefore reduce transmission of enterotoxin-producing bacteria, and ingestion of enterotoxins.     

Production of microbial rennin from Mucor miehei in a continuously fed fermenter

In this study, microbial production of rennin, a milk-clotting enzyme, from a commercial strain of Mucor miehei NRRL 3420 has been investigated in a continuously fed fermenter for prolonged times. The spherical film-type growth of the culture has been accomplished in the fermenter and the effects of medium pH, mixing and dilution rates, and feed -glucose concentration on milk-clotting activity have been elaborated. In the fermenter, optimum operational parameters have been determined as 400 rpm, 0.125 day⁻¹, and 7.5 g l⁻¹ for mixing rate, dilution rate, and feed -glucose concentration, respectively. Under these conditions, the fermenter operated 575 h continuously producing 1.24 IU ml⁻¹ maximum milk-clotting activity without concentration. In the fermenter sample at maximum milk clotting activity, the R factor and specific milk-clotting activity were determined as 1.55 × 10⁻³ IU PU⁻¹ and 5.28 IU mg⁻¹ medium protein, respectively, denoting competitive characteristics of a commercial rennet after concentration.     

The surface growth and activity ofNitrobacter

The effect of surface attachment on oxidation of nitrite to nitrate byNitrobacter was studied in batch culture, on glass coverslips, and in continuous culture on glass beads and anion exchange resin beads in an air-lift column fermenter. In batch culture, the surfaces stimulated specific growth rate, while in continuous culture, activity of attached cells was less than that of freely suspended cells. Nitrate productivity, free cell productivity, and attached cell concentration increased exponentially at the same specific rate, termed the colonization rate, and nitrate productivity was found to be a convenient estimate of biomass concentration. Permanent attachment was mediated by production of slime material. Surface growth resulted in multiple steady states and the ability to respond quickly to changes in dilution rate. The air-lift column fermenter system provided a convenient system for the study of growth and activity of attached cells and was most suitable when using ion exchange resins as a substratum for attachment.     

Analysis of secreted protein profile and enzymatic activities from Corynebacterium diphtheriae and Bordetella pertussis on production batch media using peptide quenched fluorescent substrates

Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.     

Application of mathematical models to the determination optimal glucose concentration and light intensity for mixotrophic culture of Spirulina platensis

The effects of initial glucose concentration and light intensity on specific growth rate, phycocyanin concentration and cell dry weight concentration in mixotrophic batch cultivation of Spirulina platensis using both shake flask and fermenter were investigated. Based on experimental results in shake flask culture, a number of mathematical models were constructed, and the optimal initial glucose concentration and the optimal light intensity were calculated to be S_opt=2.4471 g liter⁻¹ and L_opt=3.8632 klx. Finally, a time-dependent kinetic model for mixotrophic batch cultivation of Spirulina platensis in fermenter was also proposed. This was in good agreement with the experimental results and could be employed to predict the production of biomass and phycocyanin, and the consumption of glucose in fermenter culture.     

Antibiotic Production by Anaerobic Bacteria

Soils from aerobic and anaerobic sources were investigated for the possible presence of bacteria which produce antibiotics under anaerobic conditions of growth. The screening techniques devised for this study yielded 157 soil bacteria which, during anaerobic growth, produced antibiotic activity against aerobic test bacteria.

Studies on choice of media, presence of oxygen, and changes in antibiotic activity during growth indicated that representative strains of these bacteria produced mixtures of antibiotics. The activity was heat labile.

     

油酸促进灵芝三萜液态深层发酵的工艺研究及规模化验证

灵芝三萜是灵芝中主要的活性成分之一,前期研究发现油酸可以促进灵芝三萜液态深层发酵下的发酵合成。本研究主要对油酸促进灵芝三萜液态深层发酵的工艺进行优化,并进行3L发酵罐规模的验证。通过单因素实验考察油酸的添加方式、添加时间和添加浓度对灵芝三萜的影响,结合响应面实验,获得最优工艺条件并进行验证：在发酵第32h添加1.21%高温灭菌油酸,最高灵芝三萜含量为42.69mg/g;在发酵第7h添加1.35%过滤除菌油酸,最高三萜含量为43.38mg/g,分别比对照提高2.04倍和2.08倍。在1 000mL摇瓶中添加高温灭菌油酸和过滤除菌油酸,灵芝三萜含量分别为32.18和32.48mg/g,为对照的1.96倍和1.95倍;在3L发酵罐规模下灵芝三萜含量分别为28.66和25.13mg/g,为对照的1.62倍和1.42倍。本研究系统优化了油酸促进灵芝三萜液态深层发酵的工艺条件,并在与工业生产相对应的3L发酵罐上进行验证。该研究可为灵芝三萜的规模化发酵提供重要参考和借鉴。     

Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor

The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation.     

Construction of the genetically attenuated bacteria Bordetella pertussis devoid of dermonecrotic toxin activity and producing modified nontoxic pertussis toxin form

The recombinant modified (attenuated) bacteria A. pertussis were constructed. These bacteria contained knockout mutation of the dnt gene and produced nontoxic pertussis toxin derivative. The immunological properties of the mutant bacteria B. pertussis strain KS were studied. The recombinant bacteria B. pertussis strain KS were found to be devoid of dermonecrotic toxin activity, conserved the structure of the mutant dnt gene in condition of cultivation on selective growth media, and long-term survival in laboratory animal organism. Intranasal immunization of mice with living bacteria B. pertussis, attenuated strain KS provided protection of animals from virulent strains of the pertussis. The efficiency of the protection was comparable with protection efficiency provided by standard corpuscular pertussis vaccine OSO-3.     

The influence of pressure on the growth of methanotrophic bacteria

The growth rate and the maximum cell concentration of methanotrophic bacteria are limited by the transfer of methane and oxygen to the culture fluid. The operation under moderate pressure results in an increase in driving force for the mass transfer of both nutrients and, therefore, in a large increase in the attainable biomass concentration. Our laboratory pressure fermenter with a volume of 12 litres operates under a system pressure of up to 0.5 MPa. In this reactor a maximum productivity of 6 g biomass/lh is achieved. However, operating under moderate system pressure and exhaust gas recycling has also disadvantages because the concentrations of the gas phase components may inhibit the growth process. From the results of the laboratory fermenter we have developed kinetic models of the influence of dissolved oxygen and carbon dioxide on the specific growth rate of the methanotrophic strain GB 25. These models are the basis for processing under increased system pressure and exhaust gas recycling.