【作者】 李维新；

【导师】 郑宝东；

【作者基本信息】 福建农林大学 ， 农产品加工及贮藏工程， 2012， 博士

【摘要】 本文通过从自然发酵的枇杷酒醪中，选育出具有耐硫能力强、苹果酸-乳酸酶（MLE）活力高、发酵能力好的优良苹果酸-乳酸发酵（MLF）乳酸细菌。较系统地研究了选育的乳酸细菌的生长特性、培养基优化、产苹果酸乳酸酶（MLE）的条件及其酶学特性；建立了该菌在枇杷酒上的降酸动力学数学模型，分析了MLF对枇杷酒主要品质的影响，并初步进行了该菌在枇杷乳酸发酵果汁上的应用研究。本研究能为果酒酿造及乳酸发酵果汁提供优良MLF新菌株，并为乳酸菌的商业化应用提供理论基础与技术支持，同时还可为苹果酸-乳酸酶的固定化应用及结构功能等的进一步研究提供研究基础。本文主要研究结论如下：（1）优良MLF乳酸菌的选育研究以枇杷全汁自然发酵酒醪为菌源，以SO₂耐受能力为主要考察指标，结合耐受酒精、酸、低温的能力及MLF能力，选育出可耐受总SO₂达120.0mg/L、酒精度达13.0%（v/v）、在pH值3.0能维持生长的优良MLF乳酸菌，经中国工业微生物菌种保藏管理中心﹙CICC)鉴定，确定其为植物乳杆菌（Lactobacillus plantarum），命名为植物乳杆菌R23。植物乳杆菌R23的基因序列已上传美国国立生物技术信息中心﹙NCBI﹚数据库，GenBank登陆号为：HQ658056。高耐SO₂植物乳杆菌R23的选育，突破常规乳酸菌耐SO₂能力低的技术瓶颈，解决了低SO₂环境下MLF生物降酸而导致果酒褐变和杂菌繁殖等技术难题。（2）植物乳杆菌R23的生长特性及其培养基优化研究研究植物乳杆菌R23的适宜生长温度、pH、生长曲线，并对生长培养基进行优化，结果表明，植物乳杆菌R23属革兰氏阳性菌，兼性厌氧，适宜生长温度为30℃，pH为6.0，其细胞收获的最佳时期为适宜条件下培养21-24h。筛选出植物乳杆菌R23的无Mn2+培养基LH16，采用该培养基在30℃下培养24h的菌量可达3.40×109cfu/mL，是无Mn2+MRS培养基的两倍。植物乳杆菌R23的生长特性研究及无Mn2+培养基LH16的筛选，为该菌的高密度培养、高安全性DVS菌剂的制备提供研究基础。（3）植物乳杆菌R23产MLE条件及酶学特性研究研究了植物乳杆菌R23生长期产MLE特性及影响因素，结果表明：在对数生长期内，植物乳杆菌R23产苹果酸乳酸酶（MLE）的量及其活力随着菌量的增加而增加；产酶活力较高的时期是在细菌生长进入稳定期的4-8h内；适量的L-苹果酸和NAD⁺及厌氧发酵对其产酶和酶活力均有促进作用。采用响应面分析的方法，分别得到植物乳杆菌R23产酶量、酶活力、酶总活力与发酵温度、起始pH值、L-苹果酸浓度的数学模型，优化了植物乳杆菌R23产MLE的条件。试验结果表明：影响植物乳杆菌R23产MLE总活力的主次顺序是起始L-苹果酸浓度＞发酵温度＞起始pH值；植物乳杆菌R23产MLE的最佳培养条件为：发酵温度33.5℃、pH值6.2、L-苹果酸浓度3.8g/L，在此条件下植物乳杆菌R23厌氧发酵28h的产酶量为1.34mg/mL，酶活力为462.33u，总活力为619.52U。植物乳杆菌R23MLE的适宜酶促反应温度为24-27℃，pH值为6.0；L-乳酸和SO₂对MLE酶促反应表现出一定程度的抑制作用；NAD+和Mn2+对植物乳杆菌R23MLE的酶促反应具有明显的促进作用，NAD+和Mn2+适宜的添加量分别为1.0mM和100μM。植物乳杆菌R23苹果酸乳酸酶酶促反应的米氏方程为：1/v=0.0087×1/[s]+0.0023（R2=0.9936），米氏常数Km=3.78×10^-3（mM）。（4）植物乳杆菌R23在枇杷酒及乳酸发酵果汁中的应用研究采用二次回归旋转正交组合设计，以降酸指数（Y₁）为指标，接种量X₁、酒精度X₂、总硫浓度X₃、发酵温度X₄为影响因子，建立了植物乳杆菌R23在枇杷酒中MLF动力学模型：Y₁=83.5168+9.4207X₁-1.8707X₂-2.4874X₃+1.9457X₄。该模型具有较高的拟合度，能够准确地反映出采用植物乳杆菌R23对枇杷酒进行MLF降酸作用时各影响因素与降酸率之间的关系,该模型可以为果酒的生物降酸提供理论依据。植物乳杆菌R23对枇杷酒进行MLF，可以降低枇杷酒的总酸、提高枇杷酒的柔和度、增加酒体挥发性成分和含量，能够提高枇杷酒的品质。研究了植物乳杆菌R23在枇杷果汁中生长与产酸关系以及对有机酸和糖的代谢，结果表明,植物乳杆菌R23在枇杷汁发酵前24h是以苹果酸为主要碳源进行MLF，其发酵产酸是非生长偶联型；MLF结束后，则以糖代谢为主进行异型乳酸发酵。该研究拓展了MLF乳酸菌的应用领域，为开发果汁型乳酸发酵饮料提供研究基础。更多还原

【Abstract】 A superior malolactic fementation（MLF） lactic acid bacterium was obtained byselective breeding from natural fermented loquat wine, which with high sulfur tolerance,high MLE activity and excellent MLF ability.The growth characteristics, culture mediumoptimization, productive conditions and enzymology characteristics of malolactic enzyme（MLE） of selected lactic acid bacterium were studied, the deacidification dynamics ofloquat wine, the influence on wine quality and application in loquat juice with thebacteria were also studied. This study will provide a superior malolactic fementation lacticacid bacterium for fruit wine making or lactic acid fermentation juice, provie a theoreticalbasis and technical support for the commercial application and immobilisation of MLE ofthis lactic acid bacteriym.The results of this study were shown as follows:（1） Study on suprior lactic bacteria selective breeding.A supeior malolactic fementation（MLF） lactic acid bacterium was obtained byselective breeding from natural fermented loquat wine, which with sulfur dioxide toleranceability as the main investigation index, combined with alcohol, acid, low temperaturetolerance ability, MLF ability. The selected lactic acid bacteria was excellent with highertolerance of sulfur dioxide to120mg/L and alcohol to13%（v/v）, it could keep growingwell under pH3.0.The lactic acid bacterium was identificated as Lactobacillus plantarumby the China Center of Industrial Cuture Collection（CICC）, and was named Lactobacillusplantarum R23. Gene sequences of Lactobacillus plantarum R23had been set up a fileupload to the U.S. National Center for Biotechnology Information（NCBI） dater bank,which GenBank number was HQ658056.It had brokenthrough the conventional lactic acid bacteria resistant to low sulfurdioxide ability of the technical bottleneck with selective breeding of Lactobacillusplantarum R23with high resistance to the sulfur dioxide, and it solved the technicaldifficult problem that it will lead to wine browning and miscellaneous bacteria growthingunder low sulfur dioxide environment to MLF biological deacidification.（2） Study on growth charicteristics and culture medium optimization ofLactobacillus plantarum R23.The suitable growth temperature, pH value, growth curve and culture mediumoptimization of Lactobacillus plantarum R23were studied, the results showed thatLactobacillus plantarum R23was gram-positive bacteria, facultative anaerobic, the most suitable growth temperature was30℃and pH value was6.0, the cells harvest time wascultured21to24hours under the suitable growth condition. Culture medium of LH16without Mn2+was selected for the suitable growth of Lactobacillus plantarum R23. Byincubating the Lactobacillus plantarum R23in medium LH16at30℃for24hours, the celldensities were up to3.40×109cfu per mL, which was twice than incubated by MRS mediumwithout Mn2+. It could provide research foundation of high density culture, high securityDVS bacterium agent research and application with the study on growth charicteristics andnon-Mn+culture medium optimization of Lactobacillus plantarum R23.（3） Study on produing MLE conditions and enzymology characteristics ofLactobacillus plantarum R23.The produing MLE charicteristics during growth and effecting factors on produingMLE of Lactobacillus plantarum R23were studied, the results indicated that thecontent and activity of malolactic enzyme increased with increasing of bacteria biomassat logarithmic phage, enzyme activity reach a high value during4-8hours of stationaryphase; It showed positive correlation at bacteria growth early phage between thecontent and activity of malolactic enzyme, but showed negative correlation at laterphage.A moderate amount of L-malic acid or NAD+or anaerobic fermentation couldincrease enzyme production and activity.Response surface methodology was used to optimize conditions of MLE productionon the basis of pre-experiments, and a second order quadratic equation was finallyobtained.Results showed that the effecting order of three factors to the total activity of MLEwas as follows: concentration of L-malic acid＞fermentation temperature＞pH value.Theoptimized conditions were fermentation temperature33.5℃, pH value6.2, L-malic acidconcentration3.8g/L.Under the optiimal conditions, MLE production of Lactobacillusplantarum R23with anaerobic fermentation28hours was1.34mg/mL, MLE activity andthe total activity was up to462.33u and619.52U respectively.The optimum conditions of enzymatic reaction for Lactobacillus plantarum R23wereunder pH6.0and temperature at24-27℃. L-latic and SO₂showed against the enzymaticreaction of MLE. However, NAD⁺and Mn2+showed positive effects on the enzymereaction, which proper added conentration were1.0mM and100μM respectively.TheMichaelis equation of enzymatic reaction of MLE was:1/v=0.0087×1/[s]+0.0023（R²=0.9936）, and the Km was3.78×10^-3mM. （4）Study on application in loquat wine and lactic acid fermented juice ofLactobacillus plantarum R23.Based on quadratic orthogonal design with the deacided index （Y₁） as target,inoculation amount（X₁），alcohol degree（X₂），total sulfur dioxide concentration （X₃）and fermented temperature（X₄）as effecting factors, the mathematical model of MLF byLactobacillus plantarum R23in loquat wine was established:Y₁=83.5168+9.4207X₁-1.8707X₂-2.4874X₃+1.9457X₄。The model had a high fitting degree,It could be able to accurately reflect the relationship between the various influencing factorsand deaciding rate.Malolactic fermentation with Lactobacillus plantarum R23in loquatwine could reduce the total acid,increase the soft degree,change the volatile compositionand contents, and it could improve the loquat wine quality.The relations between growth and acid yield of Lactobacillus plantarum R23inloquat juice was studied, as well as the organic acid and sugar metabolism were alsostudied, the results showed that Lactobacillus plantarum R23take malic acid as the maincarbon source to MLF in loquat juice fermentation before24hours.It was non-growthcoupling model for fermenting to produce acid; It was abnormity lactic acid fementationwith sugar metabolism afer MLF. This study expanded the application fields of lacticacid bacteria, and it could provide research foundations for the development of lacticacid fermented fruit juice.更多还原