p.fluorescens發(fā)酵生產(chǎn)胞外多糖的結(jié)構(gòu)分析及活性評價

1、 I P. fluorescens 發(fā)酵生產(chǎn)胞外多糖的結(jié)構(gòu)分析及活性評價 摘 要 近年來， 多糖作為一種活性物質(zhì)市場需求量日益增大， 細菌胞外多糖是細菌液體發(fā)酵代謝產(chǎn)生的， 利用細菌代謝產(chǎn)糖具有周期短、 產(chǎn)量高、 質(zhì)量穩(wěn)定的優(yōu)點，是工業(yè)生產(chǎn)多糖的一種高效、 節(jié)能的方式。 不同產(chǎn)糖菌株所需要的生長條件差異很大， 經(jīng)過定向篩選的產(chǎn)糖菌株通過優(yōu)化培養(yǎng)可以不同程度的提高胞外多糖的產(chǎn)量， 這些胞外多糖一般都具有潛在的生物學活性。 本論文對實驗室

2、前期從啤酒大麥中分離篩選出的一株細菌菌株進行了研究， 發(fā)現(xiàn)其胞外多糖產(chǎn)量和性狀相對穩(wěn)定。 以胞外多糖產(chǎn)生量為指標， 對該菌液體發(fā)酵產(chǎn)胞外多糖的培養(yǎng)條件進行了優(yōu)化；采用高效凝膠滲透色譜法、氣相色譜法、酶解法、液質(zhì)聯(lián)用技術(shù)以及核磁共振技術(shù)對該胞外多糖的結(jié)構(gòu)進行了分析； 在此基礎上初步探索了該胞外多糖體外吸濕保濕性能及抗氧化活性。主要研究內(nèi)容及結(jié)果如下： 1. 對目標菌株進行形態(tài)學和分子生物學的鑒定，經(jīng)形態(tài)學特點和 16SrDNA鑒定為熒光假

3、單胞菌， 命名為 Pseudomonas fluorescens PGM37， 目前保存于中國典型微生物保藏中心，保藏號為 CCTCC NO. M2012183。 2. 通過單因素和響應面實驗相結(jié)合的方法，確定了 P. fluorescens PGM37 發(fā)酵產(chǎn)胞外多糖的最適培養(yǎng)條件為：初始 pH 7.5，搖瓶裝液量 100 mL/250 mL，接種量 5%，培養(yǎng)溫度 28℃，搖床轉(zhuǎn)速 180 rpm；產(chǎn)糖的最佳培養(yǎng)基組成為：蔗糖36.

4、2 g/L，酵母膏 3.3 g/L，NaCl 1.1 g/L，CaCl2 0.2 g/L。在上述培養(yǎng)基組分和培養(yǎng)條件下發(fā)酵 36 h， 所得產(chǎn)量為 10.1163 g/L。 通過測定發(fā)酵曲線得到該菌株產(chǎn)糖量在 48 h 時達到最高，為 11.37 g/L。 3. 對多糖提取工藝進行優(yōu)化，選擇調(diào)節(jié)發(fā)酵液 pH 為 8，2.5 倍乙醇沉淀多糖，用去離子水復溶后再次醇沉，冷凍干燥后得多糖粗品，經(jīng)過 Sephacryl S-300HR 柱層析純

5、化得到分子量均一的多糖純品， 經(jīng)過高效凝膠滲透色譜法確定胞外多糖分子量為 367.6 kDa。 4. 經(jīng)過氣相色譜分析得到該多糖由甘露糖和葡萄糖以 1:1 的比例構(gòu)成。 紅外光譜和一維核磁均顯示該糖為 β-D-吡喃型且無其他取代基團。利用專一性降解酶水解該胞外多糖，將酶解產(chǎn)物分級醇沉分離后進行 ESI-LC-MS 分析，同時結(jié)合 1D NMR 和 2D NMR 以及紅外光譜確定其結(jié)構(gòu)重復單元為 β-D-Glcp-(1, III Stru

6、cture analysis and biological activities evaluation of exopolysaccharide produced by P. fluorescens Abstract Polysaccharides have increasing market demand in recent years because of their biological activities. Bacterial

7、 extracellular polysaccharides (EPS) come from liquid fermentation of bacteria, the advantages of bacterial fermentation are short incubation period, high yield, stable quality which present a kind of high efficiency, en

8、ergy saving industrial producing method of polysaccharides. The culture conditions of bacterial strains differ from each other, and the yields of extracellular polysaccharides of directional screened strains could be imp

9、roved to different levels by optimizing the culture conditions, these extracellular polysaccharides usually have potential biological activities. The exopolysaccharide producing strain in this paper was isolated from bee

10、r barley and preserved in our lab, which was found with relatively stable yield and character. The liquid fermentation culture conditions were optimized using the extracellular polysaccharide production as an index. The

11、methods of high performance gel permeation chromatography (HPGPC), gas chromatography (GC), enzyme hydrolysis, liquid mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR) were employed to analyze the structure

12、 of the extracellular polysaccharide. Preliminary evaluation experiments of in vitro moisture retention and absorption abilities as well as antioxidant activities of the EPS were conducted. The main research contents and

13、 results are as follows: 1. The exopolysaccharide producing strain was identified as Pseudomonas fluorescens according to the morphological characteristics and 16SrDNA sequence analysis, which was named Pseudomonas fluor

14、escens PGM37, and currently preserved in China Center for Type Culture Collection with a serial number CCTCC NO. M2012183. 2. The Fermentation conditions of EPS were optimized using statistical methods single factor expe