【作者】 崔文明；

【导师】 吕加平；

【作者基本信息】 中国农业科学院 ， 农产品质量与食物安全， 2014， 博士

【摘要】 乳酸菌由于其在维持宿主体内肠道菌群平衡方面的生理功能，被认为是最安全和最具代表性的肠道益生菌，在世界范围内的发酵乳制品加工中被广泛应用。乳酸菌自溶是发酵乳制品生产中的常见现象，自溶速率决定着发酵乳制品的质量、风味及产品生产周期，在不同发酵制品中所起作用不同，有利亦有弊。因此，研究乳酸菌自溶影响因素及产生机理将更好地指导乳酸菌在工业生产中的应用并为工业生产中的实际需要选育合适的发酵菌种提供了理论依据。在此需求下，本研究从传统发酵乳制品中筛选自溶度不同的乳酸菌菌株，选取其中高自溶和低自溶度菌株各一株作为主要研究对象，研究了不同因素对高低自溶度乳酸菌自溶及各种自溶酶表达情况的影响。对低自溶度菌株，采用N+注入诱变筛选自溶度正突变和负突变菌株，采用荧光定量实时PCR检测突变前后自溶酶的表达差异，测序检测自溶酶变异位点。对高自溶度菌株，选取其中的关键性自溶酶，通过基因敲除的手段验证其在自溶中的作用。综合上述研究结果最终揭示不同自溶酶在菌株自溶中的作用。本研究所取得的主要研究成果如下：1.通过采用核酸/蛋白溶出的自溶检测方法分析7种乳酸菌的自溶度得到各菌株自溶度排序为：LJJ>SY15>LB1>AST18> LGG> LB2>116-a，筛选得到高自溶度保加利亚乳杆菌LJJ和低自溶度干酪乳杆菌116-a。两者自溶度间存在极显著差异（P<0.01）。相同自溶条件下，在自溶过程中LJJ细胞完整性遭到破坏，细胞发生不同程度得萎缩和凹陷，而且在细胞表面出现孔洞，胞内内容物也开始逐步释放至周围环境中。而低自溶度菌株116-a发生的自溶程度较小，细胞虽然发生了部分萎缩并有部分细胞内容物逸出的现象，多数细胞仍基本保持了原有细胞壁的完整性，细胞表面没有出现明显孔洞和破损。2.环境pH、温度、 SDS浓度，Triton X-100浓度和不同生长阶段可通过影响保加利亚乳杆菌LJJ中自溶酶的产量和活性大小而影响乳酸菌自溶发生的速率和程度。来自不同生长阶段的保加利亚乳杆菌LJJ细胞中，对数生长期细胞的自溶度最大，稳定期细胞的自溶度次之，衰退期细胞的自溶度最小，而衰退中后期细胞因死亡基本不再发生自溶。保加利亚乳杆菌LJJ自溶的环境最适pH范围为6.0-8.0，pH低至4.0或高至10.0时，自溶基本被完全抑制。环境温度0-50℃范围内，LJJ自溶度随温度的升高而逐渐增大，高于60℃时，自溶受到部分抑制自溶度开始出现降低。SDS的存在可以抑制自溶的发生，SDS浓度为0.05g/100mL时，乳酸菌的自溶即几乎被完全抑制。SDS亦可以抑制N-乙酰胞壁质酶的活性且与抑制自溶的趋势保持一致。Triton X-100的存在可以促进自溶的产生，且随着Triton X-100含量的增大，LJJ自溶速率越快，自溶度增大。3.不同种类乳酸菌中调控自溶的关键自溶酶存在差异。AcmA，AmiA，GlcNAcase和Dac四种自溶酶中，AcmA和AmiA在保加利亚乳杆菌LJJ中的表达量变化趋势与其自溶度的变化趋势较一致，其中AcmA的表达变化幅度最大，在不同的条件下表达量可以相差129倍之多，推测保加利亚乳杆菌LJJ中最可能的关键自溶酶为AcmA。GlcNAcase和AmiA在干酪乳杆菌中的表达量趋势与其自溶度变化趋势一致，其中GlcNAcase在不同条件下表达量相差235倍，推测GlcNAcase是干酪乳杆菌116-a中的关键自溶酶。在保加利亚乳杆菌LJJ中，环境pH的变化不会对四种自溶酶的表达产生影响，表明环境中pH的变化不是诱导菌体发生自溶的原因，而引起的自溶的变化则是由于自溶酶活性存在最适作用pH的结果。而在干酪乳杆菌116-a中，Dac在低pH下表达出现上调，高pH下表达发生下调，表明Dac的表达会受到环境pH的调控。Triton X-100不仅可以促进自溶酶的酶活，同时可以诱导AcmA和AmiA的表达。4.干酪乳杆菌116-a经过离子能量30kev N+注入诱变分别在注入剂量2.5×1015ions/cm2和2.0×1015ions/cm2处筛选得到遗传稳定性良好的最大正突变菌株2株和最大负突变菌株2株。实时荧光定量PCR分析野生菌株和突变菌株中4种自溶酶表达情况发现，4种自溶酶中，只有N-乙酰葡糖胺糖苷酶的表达量发生显著的上调或下调，最大两株正突变菌株中N-乙酰葡糖胺糖苷酶的表达量上调超过了32倍，对应的最大负突变菌株中N-乙酰葡糖胺糖苷酶下调了6倍。通过对4种自溶酶的测序比对分析突变发生位点显示，在正负突变菌株中N-乙酰葡糖胺糖苷酶和N-乙酰胞壁质酶中均存在部分碱基发生突变，导致所产氨基酸发生变化。5.通过自杀载体基因敲除策略，成功构建得到含有红霉素抗性基因的N-乙酰胞壁质酶失活的保加利亚乳杆菌突变体。将红霉素抗性基因（Emr）作为选择性标记，成功插入预敲除目的基因N-乙酰胞壁质酶基因中，与乳酸菌中自杀性载体质粒pUC19相连，成功构建得到保加利亚乳杆菌LJJ N-乙酰胞壁质酶的基因敲除组件。利用响应面法优化得到保加利亚乳杆菌LJJ的最佳电转化条件为：培养基中甘氨酸含量为0.5％，电压条件为1.5kv和电阻400。在最佳的电转化条件成功将LJJ N-乙酰胞壁质酶的基因敲除组件电转化入保加利亚乳杆菌LJJ，经含红霉素的MRS平板筛选得到N-乙酰胞壁质酶插入失活的保加利亚乳杆菌，经PCR验证后最终成功获得含有红霉素抗性的N-乙酰胞壁质酶失活的保加利亚乳杆菌突变体。6. N-乙酰胞壁质酶不仅影响着保加利亚乳杆菌LJJ的自溶，同时在菌株生理生化中也起着重要作用，会影响菌体的形态，生长速率，产酸能力和贮藏性。与野生型菌株相比基因缺失菌株生物学特性产生了以下变化：自溶度发生了显著性降低（P<0.01），37℃，24h时的自溶度下降为野生菌株自溶度的约1/2。通过扫描电子显微镜观察发现基因缺失菌株单体细胞长度由之前的十几微米增长到了数十个微米，是野生型菌株菌体单体细胞长度的4-5倍。基因缺失菌株的生长速率变慢，同时菌株所能达到的最高菌体浓度降低至OD600约在2.5附近，所能达到的最大活菌约为7.0-8.0×107cfu/mL，相比野生型菌株差了一个数量级，生物量也降低了约10%。发酵产酸速率有所降低，但所能达到的最低pH由3.89降至3.78。基因缺失菌株4℃下的耐贮藏性得到大幅提升，14天菌株存活率由0.5%提升至50%。更多还原

【Abstract】 Lactic acid bacteria （LAB）are generallly recognized as safe and the most typical representativeof probiotics. LAB occcupies a central role in fermentation process and has a long and safe history ofapplication and consumption in the worldwide production of fermented food processing because oftheir physiological functions in maintaining the balance of intestinal flora of host body.Autolysis of lactic acid bacteria is a common phenomenon in fermented dairy production and playsan important role of regulating the quality, flavors and production cycle of fermenteddairy products. Therefore, the study of the influential factors and mechanism of autolysis of lactic acidbacteria would supply the better guide for the application of lactic acid bacteria inindustrial production and provide a theoretical basis for starter breeding to suit the demand offermented dairy industry.In this study, LABs with various autolysis rate were screened from the Chinese traditionalfermented dairy products. The highest and lowest autolysis rate strains were selected as the mainresearch object. First, the effects of environmental factors on the autolysis and various autolysinsexpression of lactic acid bacteria were studied. Second, low autolysis strains was induced by N+implantation, positive mutants and negative mutants were selected on the basis of autolysis rate. Thenanalyzing expression variation of selected autolysin genes between wild type strain and mutant byfluorescence quantitative real-time PCR. Furthermore, Sequencing the selected autolysin genes to findout probable base mutant site. Third, the key autolysin of the high autolysis strain was knocked out forverifing its effect on the autolysis of LAB. Finally, the autolysis rate，morphological characteristics,and other biological characteristics was investigated between wild type strain and gene knockout strain.According to the above results, to illuminate the affect of autolysin on autolysis and reveal theinteraction among different autolysins in different LAB.The main achievements of this study are as follows:1. Through analyzing the autolysis rate of7lactic acid bacteria strains isolated from the Chinesetraditional fermented dairy products by the autolysis rate test method based on detecting nucleic acid/protein dissolution, we achieved the order of autolysis rate of7strains from high to low is: LJJ>SY15>LB1>AST18>LGG>LB2>116-a and screened high autolysis strain Lactobacillus bulgaricusLJJ and low autolysis strain Lactobacillus casei116-a. There are significant differences betweenautolysis rate of LJJ and116-a (P<0.01). Under the same autolysis conditions, the LJJ cell showedvarious degrees of atrophy and sunken and holes appeared on the cell wall surface. Intracellular contentsalso began to gradually be released to the surrounding environment as a result of the damage of cellintegrity during the autolysis process. In contrast, the low autolysis strain116-a showed slight cellautolysis. Although cell displayed atrophy and intracellular contents of little cell was released to thesurrounding environment, most of the cells still remained the original integrity of cell wall, cell surface did not appear obvious holes and damage.2. The environment temperature, pH value, SDS concentration, Triton X-100concentration anddifferent growth stages can influence autolysis rate and extent of Lactobacillus bulgaricus LJJ througheffectting on autolysis enzyme yield and activity. Lactobacillus bulgaricus LJJ cells from differentgrowth stages displayed various autolysis rate. The cells from logarithmic growth phase showed themaximum autolysis rate. The cell from stability growth phase diplayed the lower autolysis rate than thatof the cell from logarithmic growth phase. Then the cells from recession phase owned the minimumautolysis rate and cells from the mid to late of recession phase no longer occurred autolysis because ofcell death. Autolysis of Lactobacillus bulgaricus LJJ had the optimum environment pH range of6.0-8.0and when environment pH≤4or≥10, autolysis can be almost inhibited completely. In thetemperature range of0-50℃, the autolysis rate increased with temperature increase. However, when thetemperature was higher than60℃, its autolysis was partially inhibited and autolysis rate began todecrease. The presence of SDS can suppress autolysis, when the concentration of SDS increased to0.05g/100mL, autolysis of lactic acid bacteria was almost completely inhibited. SDS can also suppress theactivity of lysozyme and suppression curve was consistent with the autolysis supperssion trend. TritonX-100can enhance lysozyme activity and autolysis rate and extend of LJJ increased with content ofTriton of X-100increase.3. Autolysis of LABs was regulated by different key autolysins. Among AcmA, AmiA, GlcNAcaseand Dac, the expression trend of AcmA and AmiA was consistent with trend of auolysis rate inLactobacillus bulgaricus LJJ. The relative expression range of AcmA of LJJ from several times tohundred times, depending on different conditions.So the most likely key autolysin of Lactobacillusbulgaricus LJJ is AcmA. While The expression trend of GlcNAcase and AmiA was consistent withtrend of auolysis rate in Lactobacillus casei116-a. The relative expression range of GlcNAcase of116-afrom several times to hundred times, depending on different conditions. So the most likely key autolysinof Lactobacillus casei116-a is GlcNAcase. For Lactobacillus bulgaricus LJJ, The expression of fourautolysins were almost not influenced by environment pH value variration. The changes of cell autolysisrate with environment pH value variation were due to high autolysin activity under the optimum pHconditon rather than autolysins expression increase. Dac expression was up-regulated under low pH anddownregulated under high pH, which suggested that the expression of Dac will be affected by theenvironment pH in Lactobacillus casei116-a. Triton X-100can accelerated autolysis of LAB throughthe upregulation of AcmA and AmiA expression.4. Lactobacillus casei116-a was induced by N+implantation with energy of30Kev. Twomaximum positive mutants and two negative mutants with good genetic stability were screened underN+implantation dose2.5×1015ions/cm2and2×1015ions/cm2respectively. Real time fluorescencequantitative PCR analyzed the relative expression of aotulysins between wild type strain and the mutantstrain.The results showed only the expression of N-acetylglucosaminidase was significantlyup-regulated or down-regulated among four selected autolysins. N-acetylglucosaminidase was up-regulated exceeded30times in positive mutant while that was down-regulated6times in negaitivemutant. Sequencing four autolysin of mutants showed several base site of N-acetylglucosaminidase andN-acetylmuramidase were change and leaded to the amino acid change.5. Through suicide plasmid vector gene knockout strategies, we successfully constructed theinactivation of N-acetylmuramidase Lactobacillus bulgaricus mutant containing erythromycinresistance gene. The erythromycin resistance gene (Emr) as a selective marker, was successfully insertedinto the N-acetylmuramidase gene. Then it was connected with the suicide plasmid vector pUC19forlactic acid bacteria successfully. So N-acetylmuramidase gene knockout component was constructed forLactobacillus bulgaricus LJJ. Optimizing electroporation conditions of Lactobacillus bulgaricus LJJ byresponse surface method and the optimal electroporation conditions electric voltage, resistence andglycine content of1.5kV,400and0.5g/100mL culture medium. Under the optimal electroporationconditions, the N-acetylmuramidase gene knockout component was transformed into Lactobacillusbulgaria LJJ cell. Then N-acetylmuramidase insertion inactivation Lactobacillus bulgaricus mutant wasscreened by MRS plate containing erythromycin sucessfully and identified by PCR verification.6. N-acetylmuramidase of Lactobacillus bulgaricus LJJ not only affects the autolysis but alsoplays an vital role of other physiological and biochemical characteristics, such as the growth rate ofbacteria, acid producing capacity and storage. Compared with the wild type strain, N-acetylmuramidasegene deletion strains produced the following changes: the autolysis rate was decreased significantly(P<0.01). The autolysis rate of24h decreased to about1/2than that of wild type strain. The result ofscanning electron microscope showed the single cell length of the gene deletion mutant increased fromthe previous ten micrometers up to tens micrometers, which is several times longer than the length ofwild type strain single cell. For gene deletion strain, the growth rate decelerated, the highest cellconcentration reduced from3.5to around2.5(OD600value), the maximum viable count of bacteriawas decreased to7.0.-8.0×107cfu/mL, compared to the wild type strain the value decreased by almostone order of magnitude and biomass was reduced by about10%. Besides, althrough fermentation ratedescended, the achievable minimum pH decreased from3.89to3.78. Storage stability of gene deletionstrains were greatly improved, the14day survival rate was increased from0.5%to50%under4℃temperature.更多还原