Lyoprotective Effect Of Soluble Extracellular Polymeric Substances From Oenococcus Oeni During Its Own Freeze-Drying Process

Malolactic fermentation(MLF)is very important for the production of wine.It is mainly conducted by Oenococcus oeni which can reduce acidity,improve flavor and increase microbiological stability.Spontaneous MLF was carried out by a variety of microorganisms,which may result in problems like high level of biological amine and volatile acid.However,Oenococcus oeni starter is a good solution to these problems.Freeze-drying is usually used in the production of Oenococcus oeni starter,but bacteria can be damaged and lead to a decline in vitality in the process of freeze-drying,while the addition of freeze-drying protectant can effectively reduce bacterial damage and maintain vitality of bacteria.Extracellular polymers substance(EPS)can be secreted by many microorganisms during growth metabolism to increase the resistance of bacteria to frozen repeatedly.EPS has great potential as a freeze-drying protectant.In this study,the mechanism of EPS against freeze-drying was explored,and EPS was combined with common freeze-drying protectant to improve the survival rate of Oenococcus oeni after freeze-drying.In this paper,the culture conditions for Oenococcus oeni to produce EPS were optimized.Then,scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to observe the morphological changes in the internal and external surfaces of bacteria after freeze-drying,combining with K~+/Na~+-ATPase activity detection and GC-MS detection of bacterial fatty acids to prove the anti-freeze-drying mechanism of EPS.Finally,EPS was compounded with conventional freeze-drying protectant to obtain the formula with the highest survival rate.The main results are as follows:(1)The optimal initial pH value of EPS production was 4.3,the optimal nitrogen source was peptone with the optimal concentration of 20 g/L,the optimal carbon source was glucose with the optimal concentration of 15 g/L.Orthogonal test showed that when the medium conditions were initial pH4.8,20 g/L peptone and 15 g/L glucose,the yield of EPS of Oenococcus oeni was the highest,up to 119.7 mg/100 mL.(2)Through SEM and TEM observation,it can be found that the protectant can fully wrap the Oenococcus oeni and form a protective layer outside the cells,and EPS can form thicker protective layer than other protectants,maintaining the cell with full smooth surface,intact cell membrane.Results of both Na~+/K~+-ATPase activity test and GC-MS test showed that the treatment groups with protectant could better maintain the ATPase activity and fatty acid composition of cells,and the cell activity was significantly higher than that of the treatment group without protectant(3)The study of single factor freeze-drying protectants obtained four protectants with the best protection effect were 15%sucrose,10%trehalose,2.5%glutamate sodium,and 5%EPS,respectively.The optimum formula was 15%sucrose,15%trehalose,0.5%monosodium glutamate and 5%EPS,of which the survival rate can reach 93.28%.