| China is the wine production and consumption country, but the malolactic starters usedin our wine industry are mainly dependent on imports. There is no malolactic starter with ourown property right in china. O. oeni SD-2a isolated by our lab from spontaneous MLF wine,is a highly stress adaptive strain. It is of theoritical and practical importance to study on O.oeni stress induced resisitance to freeze-drying mechanism and develop ready-to-usemalolactic starter with our own property right.Using O. oeni SD-2a as experimental strain, the effect of different stress treatments onthe freeze-drying viability were investigated, in order to evaluate the feasibility of applicationof stress induced cross protection into preparation of malolactic starter. Membrane fatty acidmethyl ester methods were established and the correlation of membrane fatty acid andfreeze-drying viability were analyzed, in order to discuss the mechanism of stress inducedresisitance to freeze-drying in the level of membrane fatty acid. A proteomic research wascarried out to characterize and identify proteins expressed by O. oeni SD-2a under differentstress treatments after optimizing the2D gel electrophoresis conditions, in order to discussthe mechanism of stress induced resisitance to freeze-drying in the level of cytosolic proteins.Protein or enzyme genes associated with resisitance to freeze-drying characteristics werecloned and analyzed, in order to discuss the mechanism of stress induced resisitance tofreeze-drying systemically. Main results were displayed as follows:1. Growth phase, protective agents and rehydration media were the main factors toaffect the survival of O. oeni SD-2a after freeze-drying. It was found that O. oeni SD-2a cellsin the early stationary phase survived better than those in the mid-log phase afterfreeze-drying. Sodium glutamate (2.5%) was the best protectant, giving the cell viability69.5%. When freeze-dried O. oeni SD-2a was rehydrated in ATB medium, the highestviability was obtained also.2. Optimal stress treatment before freeze-drying had obvious effect on the cell viability and MLF ability. During stress treatments, O. oeni SD-2a treated with8%ethanol resulted inthe highest freeze-drying survival rate (82%), the treatment with pH3.5also notablyincreased freeze-drying viability (80.5%), both completed the MLF within8days. Comparedto control, the cell viability after freeze-drying increased by19.2%and17.7%, the MLFability increased by29%and28%, respectively.3. Five different methods on analysis of membrane fatty acid composition ofOenococcus oeni SD-2a were compared by GC/MS chromatogram, relative content of fattyacid and relative standard deviation, respectively. Method2and4gave the best results.Method2was accurate and complete, but time-consuming. Method4was rapid, but lessintegrity.4. During stress treatments, the changes of membrane fatty acids of Oenococcus oeniSD-2a were mainly reflected by U/Scyc ratio, relative content of C19cyc11and C18:lcis11. Adecrease in UFA/SFA ratio and in the C18:1relative concentration, and an increase incyclopropane fatty acids (CFA) content mainly due to the increase in C19cyc11relativeconcentration were observed in all stress shocked cells. There was a significant negativecorrelations between C19cyc11and C18:lcis11in all stress shocks. The freeze-dryingviability exhibited a significant positive correlation with the levels of C19cyc11in cold andacid shocks. The only significant positive correlations between the ability of O. oeni SD-2a toconduct malic acid degradation and membrane composition existed with C14:0in ethanolshocks.5. Different cell disruption methods, protein lysate buffer, IPG strips, and proteinloading amount were used to optimize the2D gel electrophoresis conditions of Oenococcusoeni SD-2a. When ultrasonic method were used to break cell, urea-thiourea lysate bufferwere used to extract protein, phenol/chloroform/isoamyl alcohol were used to purify protein,and the protein loading capacity was400μg, the IPG strip was pI5-8, a reference map with afirst insight into the profile of protein expression of O. oeni were provided.6. Cytosolic proteins of the strain cultivated in different growth phases, acid shocks andethanol shocks were resolved by optimizing two-dimensional gel electrophoresis.Accompanied with the analysis of mass spectrometry and bioinformatic, heat shock proteinHsp20were concluded as key protein in the mechanism of stress induced resistance tofreeze-drying in O. oeni.7. Cyclopropane fatty acid synthase gene (cfa) and heat shock protein gene (hsp) weresuccessfully cloned, and the amino acid sequence of the gene were analyzed by bioinformatic.Combined with the analysis of membrane fatty acid composition and proteomic,cyclopropane fatty acid C19cyc11were concluded as positive role and heat shock protein Hsp20were concluded as key protein, suggesting the protein might play a potential importantrole in the mechanism of stress induced resistance to freeze-drying in O. oeni. |
PDF Full Text Request