【摘要】：植物乳桿菌可以適應(yīng)惡劣的葡萄酒環(huán)境,啟動(dòng)蘋果酸-乳酸發(fā)酵,具有產(chǎn)生植物乳桿菌細(xì)菌素的能力,抑制或殺死葡萄酒中有害乳酸菌,防止葡萄酒的破敗,部分替代二氧化硫的作用,從而降低葡萄酒中二氧化硫的使用。因此篩選產(chǎn)細(xì)菌素的植物乳桿菌進(jìn)行蘋果酸-乳酸發(fā)酵,十分必要。本試驗(yàn)首先通過共培養(yǎng)誘導(dǎo)培養(yǎng)產(chǎn)生細(xì)菌素,從14株植物乳桿菌中成功篩選獲得兩株產(chǎn)細(xì)菌素的植物乳桿菌XJ25和PC520。其次,利用兩株菌基因組重測序手段,進(jìn)一步比較分析其植物乳桿菌素(Plantaricin,pln)基因座間存在的差異,獲得植物乳桿菌素pln基因座遺傳圖譜。最后構(gòu)建基因plnE和plnF的外源表達(dá)載體,誘導(dǎo)產(chǎn)生大量重組蛋白,并對(duì)其進(jìn)行初步的純化。主要研究結(jié)果如下:1)14株植物乳桿菌中,菌株XJ25和PC520抑菌范圍大,抑菌性較強(qiáng),且對(duì)醋酸菌有一定的抑制作用;植物乳桿菌中共培養(yǎng)現(xiàn)象是普遍存在的,菌株XJ25和PC520與糞腸球菌XJ26M共培養(yǎng)誘導(dǎo)產(chǎn)生細(xì)菌素。通過對(duì)7株乳酸菌(XJA2,201,205,216,509,594,542)進(jìn)行16S rRNA及recA基因的擴(kuò)增分析,鑒定7株菌為植物乳桿菌,并且RAPD分析12株植物乳桿菌隨機(jī)擴(kuò)增片段的多態(tài)性,表明了其基因組DNA的多態(tài)性。2)菌株XJ25和PC520的pln基因座結(jié)構(gòu)相似,均由6個(gè)操縱子(plnEFI、pln JKLR、plnGHSTUVW、plnABCD、plnMNOP、plnWXY)組成,其中plnABCD是三組分調(diào)節(jié)操縱子。并且菌株XJ25和PC520均存在一個(gè)新的未知功能的orf1。植物乳桿菌菌株P(guān)C520的部分基因(pln Y,pln S)等發(fā)生缺失,菌株XJ25的plnF基因編碼蛋白在14位(A-S)和42位(V-I)有兩個(gè)氨基酸突變。3)通過一步定向克隆技術(shù),引物設(shè)計(jì)與線性質(zhì)粒兩端同源片段,成功將目的片段插入質(zhì)粒中,轉(zhuǎn)化的效率很高。優(yōu)化重組蛋白誘導(dǎo)表達(dá)條件,選擇在18℃,150 rpm,IPTG濃度0.6 mM,誘導(dǎo)時(shí)間12 h,進(jìn)行擴(kuò)大培養(yǎng)。4)重組蛋白PlnE,PlnF1,Pln F2經(jīng)AKTA蛋白純化分析,超濾脫鹽等,去除部分雜蛋白,所測蛋白濃度為600-1000 ng/μl,仍需進(jìn)一步純化;純化后蛋白進(jìn)行抑菌活性檢測表明,重組蛋白PlnE和Pln F具有抑菌活性,且蛋白PlnE和蛋白PlnF之間存在協(xié)同作用。
[Abstract]:Lactobacillus plantarum can adapt to bad wine environment, start malolactic acid fermentation, have the ability to produce lactobacillus plantarum bacteriocin, inhibit or kill harmful lactic acid bacteria in wine, and prevent wine decay. Partially replace the role of sulfur dioxide, thereby reducing the use of sulfur dioxide in wine. Therefore, it is necessary to screen Lactobacillus plantarum producing bacteriocin for malic acid-lactic fermentation. In this experiment, two bacteriocin producing strains of Lactobacillus plantarum (XJ25 and PC520.) were obtained from 14 strains of Lactobacillus plantarum. Secondly, the genetic map of pln locus of Lactobacillus plantarum (Plantaricin,pln) was obtained by comparing and analyzing the difference between the two strains by means of genome resequencing. Finally, the exogenous expression vectors of plnE and plnF were constructed, and a large number of recombinant proteins were induced and purified. The main results are as follows: 1) among the 14 strains of Lactobacillus plantarum, XJ25 and PC520 have a wide range of inhibition, strong bacteriostatic, and have a certain inhibitory effect on acetic acid bacteria; The co-culture of Lactobacillus plantarum was common. The co-culture of XJ25 and PC520 with Enterococcus faecalis XJ26M induced the production of bacteriocin. Seven strains of Lactobacillus plantarum (XJA2201205216509594542) were identified as Lactobacillus plantarum by 16s rRNA and recA gene amplification, and the polymorphism of random amplified fragments of 12 strains of Lactobacillus plantarum was analyzed by RAPD. The results showed that the pln loci of XJ25 and PC520 were similar, and they were composed of six plnEFI,pln JKLR,plnGHSTUVW,plnABCD,plnMNOP,plnWXY, in which plnABCD was a three-component regulatory operon. And both XJ25 and PC520 have a new orf1. with unknown function. Some genes of Lactobacillus plantarum PC520 (pln Yappln S) were deleted. The plnF gene encoding protein of XJ25 had two amino acid mutations at 14 (A-S) and 42 (V-I) sites. 3) one step directional cloning technique was used. The primers were designed to be homologous to the linear plasmids, and the target fragments were inserted into the plasmids successfully, and the transformation efficiency was very high. The expression conditions of recombinant protein were optimized. The recombinant protein PlnE,PlnF1,Pln F2 was cultured for 12 h at 18 鈩，

本文編號(hào)：2428609