【作者】 赵文英；

【导师】 李华；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2008， 博士

【摘要】 苹果酸-乳酸发酵（Malolactic fermentation，MLF）可使新（生）葡萄酒的生青、酸涩等特点消失，达到理想的酸度平衡，微生物稳定性增强。而能够适应葡萄酒环境、较专一地进行MLF、并对葡萄酒质有增益作用的主要是葡萄酒乳酸菌中的酒酒球菌（Oenococcus oeni）。由于葡萄酒生境恶劣复杂，人工接种诱导MLF时，往往由于菌体的大量死亡，而无法成功启动MLF。究其原因有两方面，一是实验室培养获得的菌体失去了对葡萄酒环境的天然抗性，造成接种存活率下降；二是菌体在冷冻干燥过程中受到损伤，导致冻干后存活细菌数量减少。因此，对于直投式葡萄酒乳酸菌发酵剂生产说，除保证菌体的高密度培养外，更需注重培养条件对菌体接种存活率和冻干存活率影响的研究。酒酒球菌SD-2a是本实验室从山东烟台地区自然MLF葡萄酒中分离得到，并获得国家专利保护的优良菌株。深入研究酒酒球菌SD-2a的胁迫适应性反应机制，对开发具有我国自主产权的直投式葡萄酒乳酸菌发酵制剂，具有重要的理论和实践意义。本文以酒酒球菌SD-2a为受试菌材，研究不同培养基组成、酸胁迫处理和乙醇胁迫处理对菌体生长、接种存活率、MLF活性及冻干存活率的影响，以评价在培养过程中，诱导菌体发生酸、乙醇胁迫适应性反应对提高菌体存活率的有效性，为直投式酒酒球菌SD-2a发酵剂的制备提供新的研究思路和基础实验数据。此外，本文通过测定上述培养条件对菌体细胞内MLE活性、H⁺-ATP酶活性和细胞膜脂肪酸组分的影响，以期初步探讨酒酒球菌SD-2a的酸、乙醇胁迫适应性反应机制。本文得出的主要结果有：（1）与FMATB和MATB培养基相比，ATB培养基培养获得的菌体，其细胞产量分别增加了8．5％和46．2％；其接种模拟酒培养液后的存活率分别提高了20．3％和40．2％；其冷冻干燥存活率分别提高了48．5％和68．3％；不同培养基条件下，菌体生理状态测定结果表明，与FMATB和MATB培养基相比，ATB培养基培养获得的菌体，其H⁺-ATP酶活性分别增加了22．6％和72．8％；其细胞膜中C19cyc11的相对含量分别提高了10．0％和36．8％；其细胞膜U／S值分别提高了20．4％和45．2％。故ATB培养基培养所得菌体，由于自我酸胁迫反应，增强了其对葡萄酒胁迫因素及冷冻干燥的抗性，而该反应与H⁺-ATP酶活性增强和菌体细胞膜脂肪酸组分的变化密切相关。（2）与ATB和MATB培养基相比，FMATB培养基培养获得的菌体，其菌体生长速率分别增加了12．1％和94．5％，其细胞内MLE活性分别增加了33．9％和7．1％。故混合添加有葡萄糖、果糖、苹果酸的FMATB培养基，有利于菌体生长速率及MLF活性的提高。（3）通过比较酸胁迫条件下，酒酒球菌SD-2a在三种培养基中的最大生长速率和最大OD₆₀₀值，选择FMATB培养基为酸胁迫处理用培养基。在FMATB培养基条件下，酸胁迫处理降低了酒酒球菌SD-2a菌体的生长速率，且对菌体细胞产量有一定影响，其中，pH3．2处理所获菌体细胞产量约为对照的70％。但酸胁迫处理显著增强了菌体的接种存活率，其中pH3．2处理的菌体接种存活率比对照提高了近9倍。pH3．5和pH3．2处理均能使菌体顺利启动模拟酒培养液的MLF。且酸胁迫处理能显著提高菌体的冻干存活率，其中，pH3．5和pH3．2处理所获菌体的冻干存活率分别为对照的2．2倍和1．9倍。故酸胁迫处理是直投式酒酒球菌SD-2a发酵剂生产有效的胁迫处理方式，初步筛选出酸胁迫处理条件应设置在pH3．5～3．2之间。（4）酒酒球菌SD-2a酸胁迫适应性反应机制研究结果表明，酸胁迫处理诱导增强了菌体细胞内MLE活性，其中，pH3．5和pH3．2处理菌体细胞内MLE活性，分别比对照处理提高了23．7％和46．4％；酸胁迫条件诱导增强了菌体H⁺-ATP酶活性，其中，pH3．5和pH3．2处理分别是对照处理的1．22和1．39倍；酸胁迫处理明显增加了细胞膜中环丙烷型脂肪酸的相对含量，且pH3．5和pH3．2处理与对照相比显著增加了细胞膜U／S值。故菌体在酸胁迫适应过程中，细胞内MLE活性和H⁺-ATP酶活性的升高，细胞膜环丙烷型脂肪酸的聚集及U／S值增加，应与菌体接种存活率和冻干存活率的提高密切相关。（5）通过比较乙醇胁迫条件下，酒酒球菌SD-2a在三种培养基中的最大生长速率和最大OD₆₀₀值，选择FMATB培养基为乙醇胁迫处理用培养基。在FMATB培养基条件下，乙醇胁迫处理明显降低了菌体的生长速率和生长量。与酸胁迫处理相比，乙醇胁迫处理对提高菌体的接种存活率及发酵活性，效果不明显。5％乙醇胁迫条件降低了菌体对冷冻干燥的耐受性，而10％乙醇胁迫条件能诱导增强菌体对冷冻干燥的抗性。但总体来讲，乙醇胁迫处理不是直投式酒酒球菌SD-2a发酵剂生产有效的胁迫处理方式。（6）酒酒球菌SD-2a乙醇胁迫适应性反应机制研究结果表明，乙醇胁迫条件诱导增加了菌体细胞内MLE的活性，且乙醇浓度越高，菌体细胞内MLE的活性越高；但乙醇胁迫处理却显著抑制了菌体H⁺-ATP酶活性；5％乙醇胁迫处理明显增加了细胞膜总饱和脂肪酸的比例，细胞膜U／S值降低；而10％乙醇胁迫处理明显降低了细胞膜总饱和脂肪酸的比例，增加了总不饱和脂肪酸的相对含量，细胞膜U／S值增大。（7）酒酒球菌SD-2a在10％乙醇胁迫条件下，菌体的生长速率和生长量随着培养基中苹果酸含量增加而增大。菌体生理状态测定结果表明，随着培养基中苹果酸含量的增加，菌体细胞内MLE活性显著增加，且菌体细胞膜U／S值显著增加。故在10％乙醇胁迫存在的条件下，MLF途径及细胞膜脂肪酸组分的调整，能增强菌体的乙醇耐受性，提高了菌体在乙醇胁迫环境中的生长能力。（8）处于稳定前期的酒酒球菌SD-2a菌体，其冻干存活率高于处于指数中期的菌体。酸胁迫条件和培养基组分都显著影响着菌体的冻干存活率，且两因素间的交互作用极显著。（9）脂肪酸组分与冻干存活率间具有较强的相关关系，其中C16：0与冻干存活率间存在显著负相关，C19cyc11与冻干存活率之间存在显著正相关。故认为增加细胞膜中C19cyc11的相对含量是酒酒球菌抗冷冻干燥机制之一。（10）本文的创新点：初次以本土酒酒球菌菌株为试材，进行了酒酒球菌酸、乙醇胁迫适应性反应机制的研究；本文提出在酒酒球菌SD-2a培养过程中，诱导其发生酸胁迫适应性反应可有效提高菌体的存活率，该结论为该菌直投式发酵剂的制备提供了理论支持；首次对酒酒球菌抗冷冻干燥机制进行了初步研究。更多还原

【Abstract】 During the process of winemaking,malolactic fermentation (MLF) follows alcoholicfermentation.Different bacteria genera have been reported to carry out MLF in wine.Amongthem,Oenococcus oeni is recognized as the most advantageous and tolerant bacterium.MLFdeacidifies wine and results in a softer feeling in the mouth.MLF also improvesmicrobiological stability and organoleptic characteristics.However,due to very harshenvironmental conditions in the wine for bacterial survival and growth,induction of MLF hasshown failures.There are two reasons for such failures.On one hand,strains lose their naturaladaptation to survive and growth in wine,which results in low inoculation viability;On theother hand,lyophilization offers stress conditions such as freezing,drying,and concentrationstress,which diminish cell viability.Therefore,expanding interest in freeze-driedready-to-use malolactic starter cultures has placed more emphasis on developing starterproduction and preservation methods that promote high cell viability and activity.O.oeniSD-2a isolated by our lab from spontaneous MLF wine of YanTai area,is a highlystress adaptive strain.It is of theory and practical importanc e to study on O.oeniSD-2a stressadaptive response mechanism and develop ready-to-use malolactic starter cultures with ourown property right.Using O.oeniSD-2a as experimental strain,we investigated the effect ofmedium composition,acid stress treatment,alcohol stress treatment on MLF activity afterdirectly inoculated into wine-like medium and freeze-drying viability,in order to evaluate thefeasibility of application of stress induced cross protection into preparation of starter cultures,and supply basic experimental dates.In addition,from the point of intracellular MLE activity,H~+-ATPase activity and changes in membrane fatty acid composition,the paper clarifiedO.oeniSD-2a acid and alcohol stress adaptive response and cross protective responsemechanism.Main results were displayed as follows:1.The effect of 3 kinds of culture media was investigated on the direct inoculationviability,freeze-drying viability of O.oeniSD-2a.ATB medium without supplementation ofDL-malate had weak pH buffering capability.Compared with FMATB and MATB,O.oenicells cultured in ATB increased inoculation viability and freeze-drying viability.Though ATBmedium decreased intracellular MLE activity,H+-ATPase activity was the highest among 3kinds of culture media.Concerning the membrane fatty acid composition,it was observed that ATB medium increased distinctly the relative concentration of lactobacillic acid (C19cyc11)and U/S ratio in cell membrane lipid composition of O.oeniSD-2a.The increased resistance towine stressor and freeze-drying is probably a result of the cross protection conferred by selfacid stress response induced in ATB medium,which might be related with changes inmembrane fatty acid composition of O.oeniSD-2a.2.FMATB medium is mixed with glucose,fructose and DL-malate,which makes forbacterial growth and MLF activity.Compared with ATB and MATB medium,the growth rateincreased by 12.1% and 94.5% in FMATB medium,intracellular MLE activity increased by33.9% and 7.1%.3.Acid stress treatment decreased the growth rate of O.oeniSD-2a,but took a certaineffect on cell biomass in FMATB medium.Acid stress treatment increased inoculationviability,and pH3.2 treatment increased by 9 folds compared with control treatment.Thistreatment could successfully initiate and complete MLF.Acid stress treatment all increasedfreeze-drying viability,and the freeze-drying viability of pH3.5 treatment was 103%,whichwas highest survival rate.Therefore,acid stress treatment was an effective technique forpreparation of ready-to-use O.oeniSD-2a starter cultures.pH stress conditions should be setbetween 3.5-3.2.4.Acid stress adaptive response mechanisms of O.oeniSD-2a were studied;The resultsshowed that acid stress treatment increased bacterial intracellular MLE activity andH~+-ATPase activity.Membrane fatty acid composition analysis showed acid stress treatmentdistinctly increased CFAs relative concentration,and pH3.5 and pH3.2 treatment significantlyincreased membrane U/S ratio,which should closely related with enhancement of inoculationviability and freeze-drying viability.Accumulation of CFAs in membrane lips might be oneof basic substance of acid induced cross protection response mechanism.5.Alcohol stress treatment distinctly decreased bacterial growth rate and cell biomass inFMATB medium.Compared with acid stress treatment,alcohol stress treatment had littleimpact on inoculation viability.5% alcohol stress treatment decreased freeze-drying viability,and 10% alcohol stress treatment increased freeze-drying viability.However,as a whole,alcohol stress treatment was not an effective technique for preparation of O.oeniSD-2a startercultures.6.Alcohol stress adaptive response mechanisms of O.oeniSD-2a were studied;Theresults showed that alcohol stress treatment increased bacterial intracellular MLE activity,butgreatly inhibited H~+-ATPase activity,which might be related with high medium pH.Membrane fatty acid composition analysis showed 5% alcohol stress treatment distinctlyincreased SFAs relative concentration,decreased membrane U/S ratio,which might be related with lower freeze-drying viability.But 10% alcohol stress treatment distinctly decreasedSFAs relative concentration,increased UFAs relative concentration,increased membrane U/Sratio,which might be related with enhancement of inoculation viability and freeze-dryingviability.7.In the presence of 10% alcohol stress,medium composition plays an important role ingrowth rate.The results showed malic acid in medium notably increased intracellular MLEactivity,and distinctly increased membrane U/S ratio.It was assumed that MLF pathwaycould be involved in changes in membrane fatty acid profiles,which increased bacterialalcohol tolerance.8.O.oeniSD-2a cells in the early stationary phase survived better after freeze-drying thanthose in the mid-exponential phase.Acid stress conditions and medium compositionsignificantly affect bacterial freeze-drying viability,and interaction between those two factorswas also significant.The freeze-drying viability of O.oeniSD-2a cultured in pH3.5 FMATBmedium was highest.9.The correlation matrix calculated by DPS software showed the freeze-drying viabilityexhibited a significant positive correlations with the levels of C 19cyc11,and had a significantnegative correlations with the levels of C16:0,which indicated C19cyc11,rather than C16:0,could offer more tolerance ability of O.oeniSD-2a cells to freeze-drying.However,fatty acidcomposition and freeze-drying viability of O.oeniSD-2a cells were determined by the mediumpH when cells harvested.There were significant negative correlations between the mediumpH and the levels of C19cyc11,and between the medium pH and freeze-drying viability,suggesting that low growth pH stimulated the synthesis of C19cyc11,which might play animportant role in the resistance of O.oeniSD-2a cells to freeze-drying.10.The innovation of the paper was as follows:(1) it is the first time to study O.oenistress adaptive response mechanism by using a native O.oeni strain.(2) a new idea was putforward that O.oeni acid stress adaptive response could be applied into preparation ofready-to-use malolactic starter cultures,and good experimental results were obtained.(3) Thefreeze-drying tolerance mechanisms of O.oeni were studied for the first time.更多还原