无明胶布氏菌活疫苗冻干稳定剂的筛选

目的为皮上划痕人用布氏菌活疫苗筛选存活率高、无明胶冻干稳定剂。方法以冻干活菌存活率为指标,对甘油、甘露醇、蔗糖、葡萄糖、乳糖、谷氨酸钠、甘氨酸、谷氨酸、脯氨酸和硫脲等10种稳定剂通过单因素筛选法,筛选出冻干存活率高的4种单因素稳定剂成分;将4种单因素稳定剂成分进行正交试验优化,筛选出最优稳定剂组合。结果单因素试验结果显示,甘油、葡萄糖、谷氨酸钠和硫脲4种稳定剂成分冻干后活菌存活率较高,对布氏菌活疫苗具有良好保护效果。通过正交试验筛选出最优稳定剂配方中四组分的质量分数分别为甘油1.5%、葡萄糖5%、硫脲1.5%、谷氨酸钠1.0%,该配方的冻干存活率可达81.5%。结论无明胶冻干稳定剂对布氏菌活疫苗具有较好的保护作用。     

无细胞百日咳攻击菌的制备及液氮保存方法分析

为了制备无细胞百日咳效价测定用攻击菌并用液氮进行保存,对此方法进行评价。采用原代攻击菌复苏传代后测定细菌浓度,并用蛋白胨水稀释为27亿个菌/m l,分装冻存管中。放入液氮罐内,并对液氮保存的攻击菌进行相应的检测,用统计软件计算。结果显示,此方法制备的攻击菌与传统的攻击菌无显著性差异,冻后虽攻击菌的毒力有所下降,但都在100-1000/MLD的正常范围内,且冻后0、3、6、9、12月无显著性差异。同批支间差异小,CV<5%。通过此方法制备的攻击菌稳定性好,试验结果符合《中华人民共和国药典》要求,可用于无细胞百日咳的效价测定。     

保护剂对混合甲烷氧化菌剂中菌群结构的影响

[目的]明晰混合甲烷氧化菌剂制备工艺及保护剂对菌群结构的影响。[方法]通过正交实验优化保护剂配方,并结合高通量测序分析菌剂制备前后菌群结构及甲烷降解能力变化。[结果]当保护剂配比为葡萄糖浓度(单位为g/mL菌液) 2. 5%、甘油15%、脱脂乳粉10%时,菌群存活率高达112. 7%;菌群结构由冻干前的优势菌Methylocystaceae(甲基孢囊菌属)、Acidovorax(食酸菌属)等演替为Ralstonia(罗尔斯顿菌属)、Stenotrophomonas(寡养单胞菌)等,其中对甲烷降解作用的Methylocystaceae(甲基孢囊菌属)冻前丰度71. 70%降为冻后的4. 26%;甲烷降解率由冻前的76. 21%降为冻后的74. 23%。[结论]混合菌剂制备过程中保护剂作为新碳源及能源影响了菌群结构,甲烷降解率下降2. 6%。菌群对外界因素的响应灵敏,但群体行为之间的关系和机理还有待探析。     

幽门螺杆菌的保藏

常规法、冻干法保存幽门螺杆菌极为困难。兔全血或１９９加５０％胎牛血清,于－７０℃条件下保存幽门螺杆菌新鲜培养物,存活期可达６－２２个月。     

洁净室环境监测用培养基贮存效期的验证及方法学确认

目的验证洁净室环境监测用培养基的贮存效期,同时对环境监测浮游菌、沉降菌及表面微生物检测方法进行确认。方法对连续3批次贮存0、90、120 d洁净室环境监测用胰酪大豆胨琼脂(Tryptose soya agar,TSA)培养皿和TSA(L-80)接触皿进行相应菌液适用性检查(包括促生长能力和无菌检查)验证试验;并对贮存120 d TSA培养皿和TSA(L-80)接触皿分别进行环境浮游菌和沉降菌、表面微生物最长采样时间检测,然后再进行促生长能力测试。结果 TSA培养皿和TSA(L-80)接触皿贮存120 d (2～8℃90 d,25℃30 d)的适用性检查结果均符合《中国药典》2015版(三部)对培养基质量控制的要求。贮存120 d TSA培养皿进行洁净室环境浮游菌(主动采样10 min)、沉降菌(暴露采样4 h),以及TSA(L-80)接触皿进行表面微生物(接触采样10 s)检测后均具有良好的促生长能力。结论通过验证试验,确定了环境监测用TSA培养皿和TSA(L-80)接触皿的贮存效期120 d(2～8℃90 d,25℃30 d),并确认了贮存后TSA培养皿进行洁净室环境浮游菌、沉降菌检测的有效性,以及TSA(L-80)接触皿进行表面微生物检测的有效性。     

促菌生胶囊产品质量的预测方法

本文介绍的方法是根据促菌生产品在贮存期中一系列的实测数据,采用数理统计的方法,归纳总结出的一数学方程式。据此,根据产品出厂时的有关参数值来预测产品的远期质量,达到要求的可靠程度,并能找出影响产品质量的主次因素。     

单抗清除瘤细胞后人骨髓的冷冻保存研究

本实验研究了用CD10类单克隆抗体55(McAb55)与兔补体结合的方法清除骨髓中普通型急性淋巴细胞白血病抗原(CALLA)阳性细胞后骨髓的冷冻保存问题。结果表明:①清除处理对人骨髓的粒单系集落培养(GM-CFU-C)无明显影响;②经过冷冻贮存的样品的GM-CFU-C少于未经冻存的样品;③清除瘤细胞后的样品冻存后与未经清除处理的正常骨髓样品冻存后的GM-CFU-C无显著差别;④影响冷冻保存后骨髓GM-CFU-C的重要因素是冷冻速率;⑤在本实验条件下以0.5℃/min速率降温效果最佳,冻后GM-CFU-C无改变,冷冻损伤主要发生于-40℃以前,降温至-40℃或～80℃后再快速降温影响不明显。此研究为用McAb55加兔补体清除骨髓中CALLA阳性细胞后骨髓的冷冻保存提供了依据。     

基于NGS方法评估不同保存条件对粪便样本中菌群含量的影响

目的评估粪便样本的不同保存条件对肠道微生态研究结果的影响。方法设计相关实验,比较7种不同的保存方法,基于高通量测序技术比对不同时间、不同贮存温度对粪便样本DNA质量、菌群多样性及病原菌检出等结果之间的差异。结果证实了对于粪便样本采集仍建议采取即刻提取核酸或-20℃保存的方法。同时比较多种保存方法后发现,采用不同样本保存方法受到影响的菌属集中在低丰度菌属(≤0.01%),对应较高丰度的病原菌检测结果的可靠性影响较小,样本中丰度超过千分之一的病原菌检测率超过95%。结论对于肠道微生态研究建议对粪便样本采取即刻提取核酸或-20℃保存的方法。对于由于其他原因未能妥善保存的粪便样本(如常温保存48 h),仍可对丰度超过千分之一的病原菌进行检测。以上结果对于肠道菌群研究中异地所采集的粪便样本运输和贮存具有一定的指导意义。     

提高双歧杆菌活菌制剂常温贮存稳定性的实验研究

以Ｄ８５０４青春双歧杆菌为实验３菌株,对活菌制剂常温保存的稳定性问题探讨。首先对菌种进行耐温耐氧驯化试验,然后进行双歧杆菌菌体冻干过程中加入适宜保护剂试验。结果双歧杆菌活菌制剂常温保存的稳定性显著提高。经常温贮存２７０ｄ后检测,质量符合规定。     

昂立一号口服液长期保存的实验研究

目的考察昂立一号口服液的保存情况。方法将不同月份生产的昂立一号口服液在常温、4℃及37℃加速情况下计活菌数(CFU/ml)。结果实验结果表明,昂立一号口服液常温保存1年后,菌数仍有1.0×106CFU/ml以上。结论实验证明,昂立一号口服液有良好的稳定性。     

海藻糖对双歧杆菌DM8504菌株冷冻干燥保护效果的研究

本文就海藻糖对ＤＭ８５０４双歧杆菌的冻干保护效果进行了研究,考察了海藻糖浓度、保护剂溶液与菌泥混合时间对ＤＭ８５０４冻干存活率的影响,结果证实该保护剂对ＤＭ８５０４双歧杆菌有良好的冻干保护效果,优于其它保护剂。     

对回春生生产菌种DM8504菌株的研究总结报告

本文对回春生生产菌种双歧杆菌ＤＭ８５０４株的研究作了总结报道。ＤＭ８５０４林是青春型双歧杆菌的一个变种,该菌株具有明显的生态效应,对调整肠道菌群失调有明显的效果。该菌淋经过试管内、动物体内及人体内试验证明是一个理想的微生态调节剂生产菌株。通过实验和临床观察证明,该菌株对降低血内毒素,治疗肝炎等多种疾病均有明显的辅助治疗作用。该菌株是从健康人的大便标本经分离培养并加以驯化等复杂过程而获得的生产菌株。     

Storage of Pseudomonas strains degrading the surfactants

The survival rate and the destruction activity of Pseudomonas strains decomposing anionic and ampholytic surfactants were studied in the course of their storage for a long period of time. The strains were shown to remain viable and active after being freeze-dried for a year. Therefore, lyophilisation can be recommended as the main method of storage for bacterial strains decomposing the following surfactants: sulfoethoxylate, sulfonate, cyclimide, and amidobetaine.     

Sperm Preservation by Freeze-Drying for the Conservation of Wild Animals

Sperm preservation is a useful technique for the maintenance of biological resources in experimental and domestic animals, and in wild animals. A new preservation method has been developed that enables sperm to be stored for a long time in a refrigerator at 4°C. Sperm are freeze-dried in a solution containing 10 mM Tris and 1 mM EDTA. Using this method, liquid nitrogen is not required for the storage and transportation of sperm. We demonstrate that chimpanzee, giraffe, jaguar, weasel and the long-haired rat sperm remain viable after freeze-drying. In all species, pronuclei were formed after the injection of freeze-dried sperm into the mouse oocytes. Although preliminary, these results may be useful for the future establishment of “freeze-drying zoo” to conserve wild animals.     

Predicting the stability of biological standards and products

A high level of stability is essential for any biological standard and is desirable in most other biological products. It is in general impossible to observe directly the rate of degradation of a biological standard since no independent scale of measurement is available. An indirect method is therefore required. The most common approach is the accelerated degradation test in which samples are stored for a time at elevated temperatures and then compared with samples stored continuously at low temperature. The relative degradation rates are used to fit the Arrhenius equation (relating degradation rate to temperature) and hence to predict stability under normal storage conditions. Previous statistical work on this problem is reviewed and a maximum likelihood ML approach is suggested which overcomes some of the limitations of the existing methodology. The accelerated degradation test also finds wide application in the shelf-life prediction of biological products where the same statistical methods are appropriate.     

A model for longevity prediction of freeze-dried tobamoviruses

An accelerated storage test was carried out on freeze-dried samples of five variants of tomato mosaic virus – from leaf material, plant sap and purified preparation with and without protectant, at 28, 37 and 45 °C. A model for longevity prediction of lyophilized tobamoviruses was developed. The preservation of tomato mosaic virus decreased to 10% after 0.7–2.8 years in unprotected variants and after 7.5–11 years in protected samples under real storage conditions. Experimental data have confirmed the predicted values.     

Effect of carbohydrates and related compounds on the long-term preservation of freeze-dried bacteria

A selection of bacteria were freeze-dried in horse serum containing various carbohydrates and related compounds. An accelerated storage test at elevated temperatures was used to determine the long-term viability of the dried organisms with the assumption that the results of accelerated storage reflect long-term viability. The results suggest that meso-inositol, non-reducing disaccharides and certain polyalcohols are the most suitable of the compounds tested for incorporation into suspending media for use in freeze-drying.     

Design and analysis of accelerated degradation tests for the stability of biological standards II. A flexible computer program for data analysis

The accelerated degradation test is commonly used to predict the stability of a biological standard during long-term storage at low temperature. A flexible computer program is described which has been written to analyse degradation test results by the method of maximum likelihood. In addition to predicting the degradation rate at low temperature, the program furnishes estimates of statistical precision and it carried out a test of goodness of fit of the data to the assumed Arrhenius equation model.     

Impact of polyunsaturated fatty acid degradation on survival and acidification activity of freeze-dried <Emphasis Type="Italic">Weissella paramesenteroides</Emphasis> LC11 during storage

The impact of polyunsaturated fatty acid (PUFA) degradation on the survival and acidification activity of freeze-dried Weissella paramesenteroides LC11 was investigated over 90-days storage at 4 degrees C or 20 degrees C in vacuum-sealed aluminium foil or glass tubes with two water activities (a(w)=0.11 or 0.23). Colony counts, acidification activity (% lactic acid/g), linoleic/palmitic (18:2/16:0) or linolenic/palmitic (18:3/16:0) ratio by gas chromatography and 18:2 or 18:3 oxylipins by reversed phase-high performance liquid chromatography were determined. The viable cells, acidification activity and 18:2/16:0 or 18:3/16:0 ratio decreased as the storage time increased. The survival, acidification activity and 18:2/16:0 or 18:3/16:0 ratio were greatest for the freeze-dried strain held in vacuum-sealed aluminium foil at 4 degrees C. The 18:2/16:0 or 18:3/16:0 ratio decrease was correlated with the accumulation of 18:2 or 18:3 oxylipins during storage in glass tubes. Hydroperoxy PUFAs, hydroxy PUFAs, divinyl ether PUFAs and oxo PUFAs were the main oxylipins identified. A large decrease in the 18:2/16:0 or 18:3/16:0 ratio and a rapid accumulation of oxylipins during storage might be enough to cause high cell death and loss of metabolic activity. These results provide further experimental support for the hypothesis that lipid oxidation and survival or activity of freeze-dried bacteria might be related.     

青春型双歧杆菌生态制品对小鼠肝癌抑制作用的初步研究

本文观察了青春型双歧杆菌(Bif.a)对小鼠肝癌移植瘤的抑制作用。结果发现,青春型双歧杆菌在瘤细胞移植前或移植后应用均显示了抑制肿瘤生长的作用。将青春型双歧杆菌注入肤腔可激活肤腔巨噬细胞,提高其吞噬功能和非特异性酯酶活性,而加入体外培养的小鼠肝癌细胞未显示有杀伤瘤细胞作用。认为青春型双歧杆菌的抑瘤作用可能是该菌刺激了宿主的免疫活性细胞杀伤了瘤细胞,而非直接杀伤作用。