微滴喷射玻璃化的过程损伤分析及保护剂优化

微滴喷射玻璃化保存系统产生的微滴尺寸较小,在较低浓度的低温保护剂条件下即可实现玻璃化。本文采用微滴喷射玻璃化保存系统对HepG2细胞进行玻璃化保存,研究保护剂加载过程、喷射过程、接收过程及玻璃化/复温过程对细胞造成的损伤程度,并通过降低保护剂中Me2SO浓度、添加适量海藻糖来优化保护剂配方。结果表明,微滴喷射玻璃化保存各过程对细胞均有损伤,保护剂加载过程、喷射过程、玻璃化及复温过程对细胞造成的损伤较大,薄片接收过程对细胞造成的损伤小。另外,随着保护剂中Me2SO浓度的降低,低温保存后的细胞活性明显降低;保护剂浓度相同时,玻璃化保存效果较慢速冷冻效果好;适量的海藻糖能够起到增强低温保存效果的作用,过量则起到降低作用;以5%Me2SO+0.3 mol/L海藻糖作为低温保护剂玻璃化保存细胞时,细胞存活率达(92.42±0.95)%,24 h贴壁率达到(95.64±1.03)%,微滴喷射玻璃化效果最好。

动物双歧杆菌冻干保护剂配方优化及酸对菌粉存活率的影响

目的 筛选动物双歧杆菌冻干菌粉保护剂,优化冻干保护剂配方,探究菌悬液制备过程中有机酸积累对菌粉存活率的影响。方法 以菌粉中动物双歧杆菌存活率为指标,通过发酵培养、离心收集菌泥、制备菌悬液、预冻和冷冻干燥的菌粉制备工艺,通过单因素实验和正交实验优化冻干保护剂。根据单因素实验探究冻干前菌悬液制备条件和最适pH。结果 最佳保护剂组合为5.00%麦芽糊精、6.00%海藻糖、0.15%抗坏血酸、1.50%谷氨酸钠、1.00%甘油。通过对菌悬液制备过程中菌粉活菌数的研究确定菌悬液制备和冻干条件,菌悬液pH 6.5,无菌水洗涤2次菌, 4℃菌悬液融合30 min,在-80℃预冻2 h,-40℃下干燥24 h,获得的冻干菌粉活菌数为1.38×10^(12)CFU/g,菌粉最高存活率可达98.60%。结论 本研究优化后的保护剂组合可以制备高活性动物双歧杆菌菌粉,提高经济效益。

Lactobacillus paracsei L9冷冻保护剂的优化

副干酪乳杆菌L9(Lactobacillus paracsei L9)是1株具有突出益生功能及生产性能的益生菌菌株,现阶段高性能发酵剂的制备是其成功应用于工业化生产的关键技术。而液氮深冷技术是高性能发酵剂制备的有效手段,其中保护剂的应用是液氮深冷工艺的关键因素。该研究以菌种存活率作为评价指标,在已有研究基础上对液氮深冷保护剂种类进行筛选,对保护剂浓度进行正交优化,并确定菌体与保护剂最佳混合比例。结果表明脱脂乳粉、果糖、海藻酸钠及Vc四种保护剂能对副干酪乳杆菌L9起到良好的保护作用;通过正交实验优化了保护剂配方:脱脂乳粉质量分数为10%,果糖质量分数为5%,海藻酸钠质量分数为5%,Vc质量分数为0.075%,可使菌体冷冻后存活率达到(92.23±0.86)%,显著提高在冻藏过程中菌体的存活率(P<0.05),较好地优化了Lactobacillus paracsei L9发酵剂的制备技术,并为实际的生产提供了理论依据。

发光菌冻干粉保护剂及贮藏效果的研究

探究脱脂乳类复合保护剂在冷冻干燥过程中对青海弧菌Q67(V.qinghaiensis sp.-Q67)和明亮发光杆菌T3(P.phosphoreum sp.-T3)相对发光率的影响,并对保护剂进行优化;-20、4℃以及室温(16～25℃)条件下对两种发光菌冻干粉的贮藏效果进行比较研究。结果表明:明亮发光杆菌T3对冷冻干燥的抗性要高于青海弧菌Q67;3种保护剂中,以脱脂乳、蔗糖、壳聚糖混合物为保护剂的两种发光菌粉复苏30min后的发光度最高;以脱脂乳、蔗糖、壳聚糖、谷氨酸钠混合物为保护剂的青海弧菌Q67菌粉在-20℃条件下长期贮藏效果最好;而对明亮发光杆菌T3菌粉长期贮藏而言,3种保护剂在-20、4℃和室温条件下的效果均较好。这两种菌粉在30min内快速反应,表明它们在毒性物质快速检测中有着较好的应用价值。

喷雾干燥法制备鲁氏酵母发酵剂的研究

利用喷雾干燥法可批量制备产业化、规模化用量的鲁氏酵母,显著缩短发酵周期,增加收益。本研究利用YDP培养基及20 L微生物发酵罐制备鲁氏酵母菌悬液,通过研究不同生长时间鲁氏酵母及培养基残糖含量、细胞干重的变化确定最佳喷雾干燥时间。采用单因素及正交试验对保护剂及喷雾干燥条件进行优化。研究结果表明:在发酵罐培养时间27 h时,细胞干重达到最大18 g/L,此时为最佳喷雾干燥时间。保护剂最佳配比为脱脂奶粉10%、海藻糖5%、谷氨酸钠1.5%。确定最佳喷雾干燥条件为进口温度115℃,进料流量15%(蠕动泵转速占比),保护剂与菌泥比例1∶1(g/L),按照上述条件得到发酵剂的存活率达到83%以上。

Optimization of Protectant, Salinity and Freezing Condition for Freeze-Drying Preservation of Edwardsiella tarda

Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose(8.0%), skim milk(12.0%), sodium citrate(2.0%), serum(12.0%) and PVP(2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of Na Cl was 10.0, 30.0 and between 5.0 and 10.0 g L^(-1) for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at-80℃ or-40℃ for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate(79.63%–82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy(SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda.

Optimizing the Chemical Compositions of Protective Agents for Freeze-drying Bifidobacterium longum BIOMA 5920

Freeze drying has a deleterious effect on the viability of microorganisms. In front of this difficulty, the present study adopts response surface methodology to optimize the chemical compositions of protective agents to seek for maximum viability of Bifidobacterium longum BIOMA 5920 during freeze-drying. Through the compara- tive analysis of single protectant, the complex protective agents show better effect on the Bifidobacterium viability. Human-like collagen (HLC), trehalose and glycerol are confirmed as significant factors by Box-Behnken Design. The optimized formula for these three variables is tested as follows: HLC 1.23%, trehalose 11.50% and glycerol 4.65%. Under this formula, the viability is 88.23%, 39.67% higher in comparison to the control. The viable count is 1.07×10 9 cfu·g-1 , greatly exceeding the minimum viable count requirement (10 6 cfu·g-1 ).

植物乳杆菌真空冷冻干燥工艺的优化

采用真空冷冻干燥法,在高密度培养、离心制备菌泥的基础上,通过正交试验,优化植物乳杆菌冻干保护剂及冻干条件,研究不同条件下的菌体存活率。结果表明:植物乳杆菌的最佳冻干保护剂为低聚果糖5 g/100 mL、脱脂乳粉12.5 g/100 mL、半乳糖4.5 g/100 mL、乳糖3 g/100 mL、脯氨酸2.5 g/100 mL、水溶性海藻糖3 g/100 mL;最佳冻干条件为菌泥比例50 g/100 mL、预冻温度-45℃、预冻时间4 h、冻干厚度1.0 cm、真空度0.2 mbar。此条件下制备的植物乳杆菌冻干粉菌体存活率为88.74%。