乳酸菌特色资源及乳酸菌发酵剂和代谢工程技术研究课题度执行情况报告

第第页乳酸菌特色资源及乳酸菌发酵剂和代谢工程技术研究课题度执行情况报告摘要：按照计划任务，课题针对2013年度计划任务书内容进行了研究，现已完成各项任务已完成，具体完成情况如下：从俄罗斯和国内新疆和西藏地区采集样品290余份，鉴定乳酸菌1534株，使内蒙古农业大学乳酸菌菌种资源库保藏菌种资源增至4924株，包括8个属、65个种和亚种，成为中国最大的乳酸菌菌种资源库。通过比较基因组学分析，选取dnaA、pyrG和pyrB等12个持家基因，完成Lactobacillushelveticus和Lactobacillusfermentium等8个种1726株乳酸菌的多位点序列分型研究。优化了乳酸菌多位点序列分型技术，完善了新的乳酸菌分型方法，构建了8种乳酸菌菌株MLST数据库。通过比较基因组分析，确定了嗜热链球菌产生风味物质代谢途径上与乙醛生成相关的7个关键酶基因和与双乙酰生成相关的5个关键酶基因。对333株嗜热链球菌中筛选出30株产香型菌株经q-PCR定量分析发酵和后熟不同时间段，各关键功能基因的表达量与乳酸、甲酸、双乙酰和乙醛生成量的相关性研究。对333株嗜热链球菌和194株保加利亚乳杆菌，经发酵时间、黏度、蛋白水解能力、噬菌体抗性和后酸化等特性以及其发酵乳感官评价，筛选出用于发酵乳生产的L.bulgaricus19株、S.thermophilus15株。其中L.bulgaricusND02和S.thermophilusND03各项指标表现最为优良。以L.bulgaricusND02和S.thermophilusND03作为发酵剂菌株，进行了高密度培养、冷冻干燥以及直投式发酵剂的研发，其发酵剂活菌数达1.83×1011CFU/g，冻干存活率提高至84.3%。利用L.bulgaricusND02和S.thermophilusND03及益生菌L.caseizhang、L.plantarumP-8和B.lactisV9等菌株开发出活性益生菌豆乳饮料和益生菌发酵乳产品及复合菌剂等4种。完成乳球菌klds4基因组测序及比较基因组学分析；完成植物乳杆菌和短乳杆菌厌氧和有氧条件下的代谢调控，证实了短小乳杆菌有氧代谢由乳酸丙酮酸乙酸转化的新途径，而丙酮酸脱氢酶具有关键作用；对于植物乳杆菌在有氧条件下，自身过氧化氢酶的激活对细胞的存活具有强的保护作用。

关键词：乳酸菌生物多样性多位点序列分型技术发酵剂代谢工程新产品

2013AnnualPerformanceReportontheStudyofCharacteristicResourcesofLacticAcidBacteriaandItsTechnologyofStarterCultureandMetabolicEngineering

MengheBilige1KongJian2LiuWenjun1WangJicheng1

（1.InnerMongoliaAgriculturalUniversity；2.ShandongUniversity）

Abstract：Theresearchwascarriedoutinaccordancewiththescheduledtask.Manyaspectofscheduledtaskhavebeencompleted.Andthedetailedinformationwasrespectivelydescribedasfollows：290sampleswerecollectedfromdifferentregionsofRussia，XinjiangandTibetofChina.1534strainsofLABwereisolatedandidentifiedfromthesesamples.ThenumberofLABincreasedto4924intheLABCC，Thesestainsincludedeightgeneraand65speciesandsubspecies.Multi-locussequencetyping（MLST）studieswerecompletedconcerning1726strainsincludingLactobacillus（Lb.）helveticusandL.fermentiumetc.Thestudywascarriedoutbasedon12housekeepinggenesofLAB.AnMLSTtechniqueofLactobacilluswasoptimized；newtypingmethodwasimproved.MLSTdatabaseof8speciesofLABwereconstructed.Identificationof7keyfunctionalgenesofflavorcompoundsformationofS.thermophilus，and5keyenzymegenesofdiacetylproduction.q-PCRoffunctionalgenesofflavorproductionof30S.thermophiluswasconducted.Thecorrelationanalysiswasperformedforkeyfunctionalgeneexpressionandthecontentoflacticacid，formicacid，diacetyl，andacetaldehydeduringthefermentationandripeningtimeperiods.19L.bulgaricusand15S.thermophiluswerescreenedoutusedforfermentedmilkproduction.AmongwhichND02andND03werethemostexcellentstainswithgoodindicators.High-densityculture，freeze-dryingtechnologyanddirect-to-vatyoghurtculturewasdevelopedbasedonthestarterculturestainsofND02andND03，inwhichfermentviablecountofstarterculturereached1.83×1011CFU/g，andthefreeze-driedratewasimprovedto84.3%.4kindsofproductssuchasactiveprobioticsoybeanbeverage，fermentedmilkandcompoundstrainspreparationwasdevelopedbasedonthestrainsofND02，ND03andprobioticsstrainsL.caseizhang，L.plantarumP-8，andB.lactisV9.CompletegenomesequencingofLactococcusklds4andcomparativegenomicsanalysiswasdeveloped.MetabolicregulationofaL.plantarumandL.breviswasfinishedunderanaerobicandaerobicconditions.AnewwaysofmetabolismandtransformationwasconfirmedbytheaerobicmetabolismofL.brevis.Itwasshownpyruvatedehydrogenaseplaysakeyroleforself-activationofcatalasesforL.plantarumoncellsurvivalunderaerobicconditions.

KeyWords：La