乳酸菌发酵剂制备过程中VBNC状态的研究

The producing concentrated lactic acid bacteria (LAB) starter culture is the key technology of the production of fermented dairy products. However, there are a lot of LAB enter a viable but nonculturable (VBNC) state as a response to environmental stress during the preparation of starter cultures. The occurence of VBNC greatly affected the determination on the key technology of the production LAB starter culture. In this project, the several strains LAB selected from the LAB Collection Center in the Key Laboratory of Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University (IMAU), P.R.China, are used to analyze the induction and recovery conditions of LAB VBNC state, and the viable of those strains will be monitored in the process of fermentation, high density fermentation, separation, preparation vacuum freeze drying and low temperature storage. This study may help us to understand the survival rate of LAB during process from freeze-drying of starter culture to storage of yogurt, and to provide the theoretical basis for increasing the survival rate of LAB during freeze-drying, and it is expected to reduce production cost and to provides some methods for LAB industry application development.

乳酸菌发酵剂的制备是生产发酵乳制品的核心技术，但在发酵剂的制备过程中，有许多乳酸菌在不良环境下处于活的非可培养状态(Viable But Nonculturable State，VBNC)，这极大地影响了在发酵剂制备过程中关键工艺条件对乳酸菌存活性影响的判定。本研究以内蒙古农业大学乳品实验室筛选出的具有工业化应用前景的乳酸菌为研究对象，将细菌的"活的非可培养态"（VBNC）特性引入到乳酸菌发酵剂制备过程中。在通过实验确认乳酸菌VBNC态的诱导和复苏条件的基础上，以产业化应用为导向，通过非培养技术检测乳酸菌发酵剂在发酵、真空冷冻干燥、冷藏等过程中活性保持情况，准确评估各种条件及冷冻保护剂对乳酸菌存活性的影响。此研究将加深对乳酸菌发酵剂的产业化制备及其应用过程中的活菌数量的了解，对提高活菌数量、降低发酵剂生产成本、促进我国乳酸菌产业化应用的开发提供新的思路和方法。

乳酸菌发酵剂在制备过程中不可避免地会受到各种损伤，导致菌株存活率降低、活性下降，增加生产成本。如果乳酸菌在不良环境下处于活的非可培养状态(Viable But Nonculturable，VBNC)，会极大地影响在发酵剂制备和应用时对乳酸菌活性的判定。本研究以本实验筛选的具有工业化应用前景的乳酸菌为研究对象，将细菌VBNC态的特性研究引入到乳酸菌发酵剂制备过程中，通过多种条件对乳酸菌进行VBNC态诱导和复苏，观察VBNC态细胞的形态变化，初步解析其机理，最后在发酵剂制备过程中改变工艺检测其活性的保持情况。.首先成功地建立了多种能快速检测包括VBNC态在内的活性乳酸菌的非培养计数方法。通过多种条件(生理盐水，低酸，寡营养，低温等)对Lb. casei Zhang、Lb. bulgaricus ND02、Bifidobacterium V9和Lb.plantarum P8等乳酸菌发酵剂进行VBNC态的诱导，成功地获得了Lb. casei Zhang和Lb. bulgaricus ND02等的VBNC态。VBNC态与正常态细胞对比，结果显示VBNC态细胞变短变粗，且细胞表面出现褶皱，不饱和脂肪酸含量也会增加。然后对两株菌的VBNC态细胞进行复苏，结果发现升温不能将VBNC态复苏，而向液体MRS中添加营养物质或脱脂乳培养基能使复苏的细胞达到较高的活菌数，且脱脂乳培养基是VBNC态有效的复苏条件；复苏后细胞具有发酵活力，低于正常态细胞，二者发酵乳产生的风味物质有很大差别。转录组测序结果表明诱导过程中Lb. casei Zhang的5个样本的基因表达差异显著。改变Lb. bulgaricus ND02发酵剂制备过程工艺，结果发现当发酵pH值为5.7时，高密度培养基中含4%酵母粉时活菌数高于不含酵母粉的情况，但是不加酵母粉的培养基能缩短发酵时间，冻干后活菌数能达到5.7× 1010CFU/g，菌体存活率能达到55.3%，高于前者；同样，筛选出Lb. plantarum P8的最优碳源为葡萄糖；而低温保藏过程中脱脂乳保护剂能使发酵剂保持更高的活菌数。. 本项目为国内首次对工业化乳酸菌发酵剂进行VBNC态研究，使我们更加深入了解了这几株发酵剂的特性，为发酵剂制备过程中乳酸菌活性的变化，对提高发酵剂活性、降低生产成本，为我国乳酸菌发酵剂产业化开发和应用提供了一定的理论基础。