Study On Culturing Characteristics Of Oenococcus Oeni

The malolactic bacteria such as Oenococcus oeni are lactic acid bacteria which carry out a secondary fermentation (malolactic fermentation, MLF), transforming the malic acid into lactic acid in the grape wine after alcoholic fermentation. In modern wine industry, malolactic fermentation is the indispensable process for the production of both superior quality red wines and white wines.The objective of this thesis is to investigate the culture characteristics of Oenococcus oeni, a train selected and stored by our laboratory, and wishes the results would provide some practical foundations and help to determine the mass culture process method and optimal parameters of Oenococcus oeni cells, fabricating the starter agent for malolactic fermentation later.Firstly, the effects of the compositions of culture medium on the growth of Oenococcus oeni cell were investigated. Three types of culture media including ATB, MRS and tomato juice media were used for culturing Oenococcus oeni cells. It was found that the maximum of bacteria number is 14×10⁸ cfu/mL in tomato juice media; about three times of the bacteria number 2.5×10⁸ cfu/mL in MRS and 3.3×10⁸ cfu/mL in ATB. And then orthogonal experiments of culture media were carried out to determine the optimal composition for the tomato juice media. The results shown that the optimal compositon of culture is yeast 1.0 g/L, peptone 20 g/L, glucose 14 g/L, malic acid 0.05 g/L, Tween80 1.0 g/L, diammonium hydrogen citrate 1.0 g/L, MgSO₄·7H₂O 0.1 g/L, MnSO4·H2O 0.001 g/L, cysteine hydrochloride 0.01 g/L, tomato juice25%. It was found that the optimal culture media is named as tomato juice culture.Secondly, the culture conditions of Oenococcus oeni in shaken flask were investigated. The results presented that optimal condition is pH 4.8 for culture media, 28℃, 100 r/min for rotation. And 5% inoculums pre-culturing 48h are satisfied.Thirdly, the batch culture kinetics of the Oenococcus oeni was investigated and modeled. The setup of the kinetics for Oenococcus oeni considered the substrate (glucose) comsuption, cell growth, lactic production both by gluscose (lactic fermentation) and malic acid through malolactic fermentation. The kinetic experiments of three different initial glucose concentration (15, 13, 12g/L) kinetics experiments were carried out and the kinetic parameters were determined by fitting and optimization with Matlab. Both the results and simulated values coincided very well. And the simulated results indicated that the formation of lactic acid by Oenococcus oeni under this condition is well associated with the growth rate of cells. Optimization kinetic parameters have been successfully used to predict the cultruring processes with another run of initial glucose concentration of 14g/L.Forthly, preliminary experiments of high dense culture through fed batch glucose solution were carried out. The experimental results showed that the optical density of Oenococcus oeni could reach about 27.5×10⁸ cfu/mL, two times higher than normal batch culture, but the culture time is three folds of normal batch culture. Either Feding pure glucose solution or glucose solution with trace elements Mn²⁺ or Zn²⁺ has no obvious difference on cell density. It indicted that the fed batch culture is not a good methed to increase culture density, at least in our conditions.Fifthly, the tolerability of Oenococcus oeni to pH, SO₂ and alcohol was tested to detect its adaptability in the wine. It can be concluded that pH, SO₂ and alcohol concentration would inhibit the cell's growth dramatically all. When pH of media with alcohol decreased from 3.4 to 3.2, the opitical density decreased two times, and when the alcohol concentration increased from 10% to 14%, the OD600 decreased about three times. And as the concentration of SO2 increased from 20mg/L to 50mg/L, cell concentration has about four times reduction.At last, the ability of the Oenococcus oeni transforming malic acid to lactic acid was studied inoculating it stimulant wine culture. The result of indexes proved that Oenococcus oeni could transform malic acid to lactic acid.