乳杆菌喷雾干燥过程菌体损伤与控制机制的研究

Lactobacillus is the main component of lactic acid starter and probiotics, its activity directly infect the production performance. Compared to freez-drying, spray driving is highly efficient, low-cost and more environmental friendly which becomes a highly concerned research field during the transition of lactobacillus drying. So far, a present problem for industry application is that the culture has a very low survivability and activity due to the fast dehydration during spray drying. The lactobacillus has even low survivability than the galactococcus. Based on the above, we would like to investigate the spray drying of several Bulgaria lactobacillus strains for further dairy starter cultures and probiotics preparation. The samples will be taken via online in-situ sampling technique. The water content, water activity, number of living bacterium and fermentation activity of dried samples will be analyzed to determine the critical point of drying from which bacteria survivability and activity will be affected. Subsequently the cell morphology, cell membrane, cytoplasm and related enzyme changes before and after the critical point drying will be performed to check the cell damage. Transcriptional omics, proteomics and metabolomics technology will be applied to compare the gene expression, proteins and metabolic pathways to find the key enzymes and genes which affects bacteria activity. The mechanism of bacteria damage before and after the critical point drying will be elucidated. Later on, we aim to investigate the key parameters to control the bacteria damage with dry domestication and exogenous protection technology. And this will supply a theoretical guidance for production of highly active lactobacillus as probiotics.

乳杆菌是乳酸发酵剂和益生菌制剂的重要主体，其活性直接影响生产性能。相对冷冻干燥，不断完善的喷雾干燥因具有高效、低成本和低能耗等优点而成为乳酸菌干燥方式转变的研究热点。目前喷雾干燥过程介质快速失水导致菌体存活率和活力偏低，限制了在乳酸菌活性制剂生产中应用。鉴于喷雾干燥过程乳杆菌比乳球菌存活率更低，本课题拟以多株保加利亚乳杆菌等重要乳品发酵剂和益生菌制剂乳杆菌为研究对象，采取在线原位取样技术，分析干燥样品水分含量、水分活度、活菌数和发酵活力，确定影响菌体存活和活力的干燥临界点；研究临界点前后细胞形态、细胞膜、细胞质以及相关酶变化，确定细胞损伤位点；利用转录组学、蛋白组学和代谢组学技术比较研究临界点前后菌体基因表达、蛋白质及代谢途径差异，确定影响菌体活性关键基因或关键酶，揭示菌体损伤机制；利用干燥驯化和外源保护技术对损伤进行控制研究，为利用喷雾干燥技术高效生产高活力乳杆菌活性制剂提供理论依据。

喷雾干燥具有低成本、高效快速、可连续生产的干燥技术，但热致死效应导致干燥后乳酸菌的存活率较低深入开展乳酸菌在喷雾干燥过程中的损伤机制研究将有助于保护乳酸菌活性。.本研究建立了一种研究喷雾干燥过程中不同阶段乳酸菌损失机制的取样方法，利用该取样方法可准确可靠地获得喷雾干燥不同干燥阶段的乳酸菌样品。通过建立在120/70 ℃条件下喷雾干燥过程中sp1.1活性、生理状态与水分之间的关系，揭示了喷雾干燥过程中乳酸菌损伤机制。结果表明90%的乳酸菌死亡发生在水分含量< 23.52%（水分活度< 0.82）。细胞膜的通透性发生变化，导致细胞大量死亡，是干燥过程中关键性损伤位点；在干燥之后，细胞膜发生相变，细胞壁修复功能受到抑制。乳酸菌结构损伤顺序呈从外到内的特点。蛋白质组学结果表明，喷雾干燥的损伤与物质的运输有关，Ca++/Mg++- ATP酶可能是损伤的关键。提高出口温度会降低乳酸菌喷雾干燥过程中的关键水分含量，出口温度为60 ℃时，乳酸菌在水分含量低于27.50%左右大量死亡；当出口温度为70 ℃时，乳酸菌大量死亡时的关键水分含量降至21.99%。.同时，根据乳酸菌在喷雾干燥过程中损伤顺序由外到内的特点，利用碱性蛋白与乳酸菌、酸性蛋白之间的静电吸引力，以及谷氨酰转氨酶（TGase）对蛋白质分子交联作用，缩短蛋白质与菌体之间的距离以提高细胞外的保护。结果表明牛乳碱性蛋白质（MBP）和不完全TGase处理都可以提高喷雾干燥后乳酸菌的存活率。TGase处理的MBP-大豆分离蛋白质(SPI)会显著降低喷雾干燥对菌体细胞膜的损伤程度，将存活率提高至84.78%。最后，通过电穿孔技术将海藻糖负载到细胞内以加强对细胞膜的保护。结果显示当胞内海藻糖含量> 3.47 μg/107 CFU时开始起到保护作用；当胞内海藻糖时含量达到10.09 μg/107 CFU时喷雾干燥后乳酸菌存活率可达到99.59%。