【摘要】：微生物多糖具備良好的物理性質(zhì)及生物活性,且生產(chǎn)周期短,能利用規(guī)模化深層發(fā)酵實現(xiàn)工業(yè)化生產(chǎn),具有廣闊的工業(yè)用途。因此開發(fā)性質(zhì)優(yōu)良的新型微生物胞外多糖具有重要意義。出芽短梗霉(Aureobasidium pullulans)合成的中性普魯蘭多糖性質(zhì)優(yōu)良,能在多領(lǐng)域廣泛應(yīng)用。除此之外,出芽短梗霉還能合成酸性多糖。本實驗室選育保藏有一株A.pullulans G16,能同時合成普魯蘭多糖和酸性多糖。本課題從A.pullulans G16的發(fā)酵液中分離出酸性多糖,對其純化后進(jìn)行結(jié)構(gòu)和性質(zhì)分析,并且研究了三種氮源對A.pullulans G16合成胞外多糖的影響。首先經(jīng)過乙醇沉淀、酶法-Sevag法結(jié)合脫蛋白、十六烷基三甲基溴化銨沉淀等步驟從A.pullulans G16發(fā)酵液中分離酸性多糖。再采用乙醇分級沉淀、DEAE-52纖維素柱層析等純化方法得到A.pullulans G16酸性多糖的主要組分。通過Sephadex G-200葡聚糖凝膠柱層析、紫外全波段掃描對純化后的A.pullulans G16酸性多糖進(jìn)行純度鑒定,結(jié)果表明,經(jīng)過純化的酸性多糖為均一組分。通過高效凝膠滲透色譜法得到A.pullulans G16酸性多糖的重均相對分子質(zhì)量為495450。綜合化學(xué)分析法、氣相色譜法、紅外光譜法、高碘酸氧化、Smith降解、部分酸水解法、糖苷酶酶解法及液質(zhì)聯(lián)用等分析結(jié)果可得,該酸性多糖是一種含有12.76%甘露糖醛酸的吡喃雜多糖,主要由甘露糖、葡萄糖、半乳糖按摩爾比2.52:27.21:0.74組成。該酸性多糖的主鏈由甘露糖、葡萄糖、半乳糖組成,摩爾比為2.48:15.92:0.72,其中以β-(1→3)糖苷鍵連接的甘露糖及葡萄糖摩爾比為1.56:12.80,其余甘露糖、葡萄糖及半乳糖以α-(1→4)鍵連接。該酸性多糖的側(cè)鏈由β-(1→6)糖苷鍵連接的葡萄糖組成。多糖的性質(zhì)研究表明,A.pullulans G16酸性多糖溶解性較差,微溶于去離子水、NaOH與HCl溶液,不溶于DMSO、乙醚與丙酮。該酸性多糖水溶液表觀粘度較大,且隨溶液濃度增大而增加;在30-80℃以及pH 4.0-10.0范圍內(nèi),溶液表觀粘度穩(wěn)定。該酸性多糖溶液表現(xiàn)出一定耐鹽性,K+和Ca2+對其溶液表觀粘度無明顯影響。在0-1mg/m L濃度范圍內(nèi),A.pullulans G16酸性多糖的還原能力以及對羥基自由基、超氧陰離子自由基、DPPH自由基的清除率隨溶液濃度增大而增加,該酸性多糖對上述自由基清除率的IC50值分別為1.45 mg/mL、3.62 mg/mL及1.11 mg/mL。研究三種氮源對A.pullulans G16合成胞外多糖的影響,結(jié)果表明,(NH4)2SO4和高濃度(≥1.8 g/L)酵母粉有利于A.pullulans G16合成普魯蘭多糖,而NaNO3和低濃度(1.8 g/L)酵母粉則更有利于A.pullulans G16合成酸性多糖。
[Abstract]:Microbial polysaccharide has good physical properties and biological activity, and its production cycle is short. It can be used in large-scale submerged fermentation to achieve industrial production, and has broad industrial use. Therefore, it is of great significance to develop a new type of microbe exopolysaccharide with excellent properties. The neutral pullulan synthesized by (Aureobasidium pullulans) has good properties and can be widely used in many fields. In addition, it is also able to synthesize acidic polysaccharides by Phyllostachys communis. A strain of A.pullulans G16 was selected and preserved in our laboratory, which can synthesize both pullulan polysaccharide and acid polysaccharide simultaneously. In this paper, acidic polysaccharides were isolated from the fermentation broth of A.pullulans G16, and their structures and properties were analyzed after purification. The effects of three nitrogen sources on the synthesis of extracellular polysaccharides from A.pullulans G16 were studied. At first, acidic polysaccharides were separated from A.pullulans G16 fermentation broth by ethanol precipitation, enzymatic-Sevag deproteinization and cetyltrimethylammonium bromide precipitation. The main components of A.pullulans G16 acid polysaccharide were obtained by ethanol fractionation, DEAE-52 cellulose column chromatography and other purification methods. The purified acidic polysaccharide of A.pullulans G16 was identified by Sephadex-G _ (200) dextran gel column chromatography and UV-full-band scanning. The results showed that the purified acid polysaccharide was homogeneous. The average molecular weight of A.pullulans G16 acid polysaccharide was 495450 by high performance gel permeation chromatography. The results of chemical analysis, gas chromatography, infrared spectroscopy, periodate oxidation, Smith degradation, partial acid hydrolysis, glucosidase hydrolysis and liquid mass spectrometry were obtained. The acidic polysaccharide, which contains 12.76% mannitonic acid, is mainly composed of mannose, glucose and galactose in the molar ratio of 2.52 to 27.21 脳 0.74. The main chain of the acidic polysaccharide was composed of mannose, glucose and galactose, and the molar ratio of mannose to glucose was 2.48 脳 15.92 and 0.72, in which the molar ratio of mannose to glucose connected with 尾-(1) glycoside bond was 1.56% and 12.80, respectively. The remaining mannose, glucose and galactose were linked by a-(1)-4 bond. The side chain of the acid polysaccharide consists of 尾-(1)-glucoside-bonded glucose. The results showed that A.pullulans G16 acid polysaccharide had poor solubility and was slightly soluble in deionized water, NaOH and HCl solution, and insoluble in DMSO, ether and acetone. The apparent viscosity of the acidic polysaccharide aqueous solution is larger and increases with the increase of the concentration of the solution, and the apparent viscosity of the solution is stable in the range of 30? 80 鈩，

本文編號：2437129