基于灌流浓缩培养的罗伊氏乳杆菌高密度发酵研究

High cell density fermentation of Lactobacillus reuteri based on concentrated fed-batch technology

罗伊式乳杆菌作为美国食品药品监督管理局(Food and Drug Administration,FDA)和国家卫生部批准的食用级益生菌,具有重要的益生功能,已被广泛地应用于食品与发酵行业,因此,研究其高密度发酵具有重要的意义。该研究首先探索了批培养、批补料培养和灌流浓缩培养对罗伊氏乳杆菌发酵生物量的影响,然后以灌流浓缩培养方式探究发酵过程中培养条件对发酵液中活菌数的影响。结果表明:灌流浓缩培养相比于批培养和批补料培养,能极大地延长菌株对数生长期的持续时间,活菌数高达2.98×10^(15)CFU/mL,菌体干重较前两者分别提高了5.3和3.6倍;在通气(氧含量恒定设定为15%)、恒pH为5.5、流速40 mL/min的条件下灌流培养,活菌数最高达8.1×10^(15)CFU/mL,菌体干重为60.96 g/L,相比于未通气和不维持恒定pH值的发酵方式,菌体干重分别提高了2.72和1.07倍。综上结果表明,灌流浓缩培养方式能实现罗伊氏乳杆菌高密度发酵,活菌数和产量都凸显出巨大的优势,该研究为罗伊氏乳杆菌的高效制备奠定了较好的基础。

Lactobacillus reuteri,as a potential probiotic approved by Food Drug Administration and Chinese Ministry of Public Health,has been widely used in food and fermentation industries.Thus,it is of remarkable importance to develop the strategy for its high-cell-density(HCD)fermentation.In this study,the effects of batched culture,batch-fed culture and concentrated fed-batch(CFB)on the biomass of L.reuteri were compared.Then,the number of viable bacteria with or without aeration and with or without maintaining a constant pH were studied during CFB fermentation.The results showed that tangential perfusion and CFB can greatly prolong the logarithmic duration of the strain,and the number of viable bacteria was as high as 2.98×10^(15) CFU/mL.Compared with batched culture and batch-fed culture,the cell dry weight(CDW)in CFB fermentation increased by 5.3 times and 3.6 times,respectively.Under the conditions of ventilation(with a stable oxygen level of 15%),a constant pH level of 5.5 and a flow rate setting at 40 mL/min in CFB fermentation,the number of viable bacteria could reach up to 8.1×10^(15) CFU/mL,with a final CDW of 60.96 g/L which showed an improvement by 2.72 times and 1.07 times in comparison to the culturing manners of non-aeration and non-maintenance of constant pH,respectively.Together,these results suggest that HCD fermentation of L.reuteri can be achieved by a specific strategy of tangential perfusion and CFB,with considerable advantages in terms of obtaining live bacteria and biomass,laying the groundwork for a low-cost and time-saving large-scale production of L.reuteri.