The Study On Liquid Nitrogen Storage Of Oenococcus Oeni

At present, liquid nitrogen cryopreservation have been used to conserve multiple microbes for long time successfully, such as alga, protozoa, yeast, lactic acid bacteria, iron bacteria, sulphur bacteria, et al. But there are few researches on the Oenococcus oeni storaging in liquid nitrogen. In this paper, we describe a cryopreservation protocol for the cultures of Oenococcus oeni. We investigated the influence of the age, rate of freezing, the temperature of thawing, their concentration in the protective medium, and the protective medium on the number of viable cells after cryopreservation. Besides, the growth characteristics, vinification characteristics and enzyme activity were also studied. The main results are listed as follows:1. Cryopreservation conditions for Oenococcus oeniWe developed an efficient cryopreservation technology for Oenococcus oeni. We got the highest number of viable cells after cryopreservation by collecting cells in the early stationary growth phase, keeping their concentration in the protective medium at 10⁹ CFU/mL, transferring cells into liquid nitrogen directly and thawing at 37℃in the water bath.2. The influence of cryopreservation period to the ratio of viable cellsThe ratio of viable cells was above 99% for 23 of the Oenococcus oeni after storaging in liquid nitrogen for 8 months, and the ratio of viable cells for sx^-1b also reached 97.1%. But the ratio of viable cells was 11.9% for Oenococcus oeni after storaging in glycerin, the hinghest ratio of viable cells was only 26.9%.3. The influence of cryopreservation to the growth rate of Oenococcus oeniAfter storaging in liquid nitrogen, the growth rate of Oenococcus oeni had no obvious change, the cell grow rate increased a little after cryopreservating in liquid nitrogen than before cryopreservation. The pH decreased as the cell grew, and the lowest pH was about 3.3. Oenococcus oeni SD-2a after storaging decreased the medium pH faster than before storaging. When Oenococcus oeni SD-2a before storaging cultured in ATB for 12,36,60,84 hours, the OD and medium pH were 0.100,4.80;1.459,3.85;2.180,3.49;2.240,3.31 respectively. And Oenococcus oeni SD-2a after storaging cultured in ATB for 12,36,60,84 hours, the OD and medium pH were 0.150,4.79;1.573,3.77;2.180,3.41;2.240,3.30 respectively.4. The influence of cryopreservation to the ability of consuming malic acid of Oenococcus oeniAfter cryopreservation,all of 23 Oenococcus oeni retain the initial vinification characteristics. Oenococcus oeni SD-2a before and after cryopreservation consumed 1.74g/L and1.75g/L malic acid respectively; Oenococcus oeni 31-DH consumed 1.36g/L and 1.66g/L malic acid respectively; and malic acid consumption by the others before and after cryopreservation were also similar. After cryopreservation,volatile acid content of the wine were under 1.2 g/L acetic acid, so the wine were healthy.5. The influence of cryopreservation to the enzyme activity of Oenococcus oeniAfter storaging in liquid nitrogen, Oenococcus oeni SD-2a intracellular MLE activity and H⁺-ATPase activity were as high as before storaging. Before cryopreservation, the MLE activities of SD-2a at early logarithmic phase, middle logarithmic phase, early stationary phase and middle stationary phase were 60.00, 170.00, 249.00, 158.00μmol.malate.h^-1mg^-1protein. And after cryopreservation, the MLE activities of SD-2a at early logarithmic phase, middle logarithmic phase, early stationary phase and middle stationary phase were 55.00, 171.00, 251.00, 156.00μmol.malate.h^-1mg^-1protein respectively. Before cryopreservation, the H⁺-ATPase activity of SD-2a at early logarithmic phase, middle logarithmic phase, early stationary phase and middle stationary phase were 4.25, 12.24, 18.21, 10.49μmol.Pi.h-lmg-lprotein respectively; after cryopreservation, the H⁺-ATPase activity of SD-2a at early logarithmic phase, middle logarithmic phase, early stationary phase and middle stationary phase were 4.13, 12.63, 17.94, 10.95μmol.Pi.h-lmg-lprotein respectively.