【作者】 刘晓永；

【导师】 王强；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白， 2007， 博士

【摘要】 β-D-葡聚糖存在于许多细菌、真菌和高等植物中,它的一个重要来源是酿酒酵母（Saccharomyces cerevisiae）细胞壁。大量研究表明酵母β-D-葡聚糖可以通过激活巨噬细胞以起到抗肿瘤、抗菌、愈合伤口、抗氧化和降血脂等功效。此外,酵母β-D-葡聚糖在食品工业中被广泛用作于增稠剂、乳化稳定剂和脂肪替代品。目前国际上还没有酵母β-D-葡聚糖的标准测定方法。常规酵母β-D-葡聚糖的测定一般采用苯酚-硫酸法,测定结果误差较大。在此,本研究建立了一个酵母β-D-葡聚糖测定的新方法:首先利用涡流微珠破壁法对酵母细胞进行破壁,酵母细胞破壁率可达到95.28%;接着采用高浓度酸预处理结合常规酸水解方法对酵母β-D-葡聚糖进行酸水解,酵母β-D-葡聚糖的酸解回收率高达98.76%;在此基础上,由GOPOD法测定出β-D-葡聚糖的含量。新方法的相对标准差和加样回收率分别为0.46%和99.96%。采用新方法对不同酿酒酵母菌种的β-D-葡聚糖含量进行了分析对比,结果表明酵母中β-D-葡聚糖含量随其菌种不同而有明显差异（P≤0.05）,占细胞壁干重比率的最大差异为138.10%,而占细胞干重比率的最大差异高达189.52%。接下来对酿酒酵母的发酵条件进行了优化。首先进行了单因素实验的研究,并在此基础上,以二次正交旋转组合设计试验,得到培养基模型方程为: Y = 106.89+3.74X₁-8.85X₁²-5.72X₂²-7.96X₃²-7.52X₄²,经二次多项式逐步回归分析确定了最佳培养基（/100ml）为:葡萄糖3.27 g、蛋白胨（2）1.89 g、酵母膏1.57 g和甘油1.04 g。经过培养基的优化后,酵母β-D-葡聚糖产量由原来的65.80 mg/100ml提高到109.33 mg/100ml。然后,采用正交优化方法进行发酵条件优化,确定主次影响因素依次为:装液量>温度>起始pH>接种量,最适培养条件为:pH5.0、接种量5ml/100ml、温度32℃、装液量60 ml/瓶,经试验验证β-D-葡聚糖量达到128.30 mg/100ml,较发酵条件优化前提高了17.35%。在发酵条件优化基础上,以Logistic、BoxLucas1和SRichards2三种模型对酵母菌生物量和β-D-葡聚糖生成量进行非线性拟合,确定生物量模型以Logistic模型拟合效果最佳,拟合方程:相关系数可以达到0.975,而在β-D-葡聚糖生成模型拟合中,以SRichards2模型最佳,拟合方程:相关系数为0.996;在对基质（葡萄糖）的消耗拟合中,采用Exponemtial ExpDec1模型,拟合方程:相关系数为0.977。传统酵母β-D-葡聚糖的制备主要采用酸碱法,这易使β-D-葡聚糖发生氧化降解,致使得率偏低,并且会对β-D-葡聚糖的天然构象部分地破坏而使其生理活性受到严重的影响。基于此,本文建立一个由酿酒酵母中分离提取β-D-葡聚糖的新方法,此方法是在较为温和的条件下进行的,包括酵母的诱导自溶、高温和有机溶剂的抽提、均质破壁、生物酶解等步骤。新方法制备的β-D-葡聚糖纯度高达93%,得率为酵母细胞干重的11%。酵母β-D-葡聚糖不溶性制约其在工业中的应用,本研究采用超声修饰和均相硫酸酯化技术对酵母β-D-葡聚糖进行修饰改性研究。以400W的功率对酵母β-D-葡聚糖进行超声处理,每循环处理时间24s,间歇时间6s,共20个循环,酵母β-D-葡聚糖的平均粒径由起始的56.49μm降到2.33μm;在Urea-DMSO的均相体系中,以硫酸为酯化剂,对酵母β-D-葡聚糖进行均相硫酸酯化反应,硫酸浓度5%,100℃下反应4 h,最终酯化β-D-葡聚糖的取代度和得率分别为0.43和87.97%。采用了FTIR、EA和NMR等方法对酵母β-D-葡聚糖及其硫酸酯的基本结构进行分析,确定酵母β-D-葡聚糖以β-（1,3）为主链,其中新方法制备β-D-葡聚糖以β-（1,3）为主链每九个单元与一分子葡萄糖通过β-（1,6）键连接,而酸碱法制备β-D-葡聚糖以β-（1,3）为主链每五个单元与一分子葡萄糖通过β-（1,6）键连接;酵母β-D-葡聚糖硫酸酯化学通式为（C6H10O5）20·9SO3·20H2O,硫酸基团取代位置在糖环的C-6上。此外,本研究还采用激光光散射（MALLS）技术结合YFY和KP蠕虫模型的方法进行研究。判定酵母β-D-葡聚糖硫酸酯溶液构象为一种介于柔性链和刚性链之间的半刚性链构象,并得到其分子层面的构象参数:ML = 646 nm-1,q = 5.1 nm,d = 0.99 nm,C∞= 16.33。最后通过动物试验,证实酵母β-D-葡聚糖及其硫酸酯具有增强免疫功效,其中新方法制备酵母β-D-葡聚糖对Con A诱导小鼠淋巴细转化率具有极显著性差异（P≤0.01）,对小鼠NK细胞活性具有显著性差异（P≤0.05）;酸碱法制备酵母β-D-葡聚糖对Con A诱导小鼠淋巴细转化率和小鼠NK细胞活性具有显著性差异（P≤0.05）;而经超声处理和硫酸酯化之后的酵母β-D-葡聚糖衍生物对Con A诱导小鼠淋巴细转化率、小鼠血清中总补体溶血活性（CH50）和小鼠NK细胞活性均具有极显著性差异（P≤0.01）。更多还原

【Abstract】 β-D-Glucans, homopolymers of glucose, was widely distributed in the cell walls of microorganisms, mushrooms and plants. One important source ofβ-D-glucans was the cell wall of yeasts, particularly of the baker’s and brewer’s yeast Saccharomyces cerevisiae. Since the 1940s, numerous studies have demonstrated thatβ-D-glucans could enhance innate host defenses by binding to specific macrophage receptors and activating macrophage, resulted in antitumour, antibacterial, and wound-healing activities. Moreover, yeastβ-D-glucans was widely applied in the food industry as a thickening and water-holding agent, emulsifying stabilizer and fat replacer. The mensuration way for detectingβ-D-glucan was a difficult problem and there had no a standard mensuration way in the world. Routine mensuration way for detectingβ-D-glucan was phenol-sulfated acid method, but the method was easily to be disturbed by other polysaccharides and lead to receive incorrect results. In our research, a new method for quantitative determination of yeastβ-D-glucan was proposed. Firstly, vortex-mini-bead disruption way was used to broke the yeast cell walls and the ratio of broken yeast cells reached 95.28%; Secondly, modified sulfuric acid hydrolysis method was adopted to completely release glucose and the recoveries of hydrolysis ofβ-D-glucan came to 98.76%; Based on this, using GOPOD enzyme way, the content of the yeastβ-D-glucan was obtained. The new the relative standard deviation value and the average recovery of the new method were 0.46% and 99.96%, respectively. The ratios ofβ-D-glucan in various strain Saccharomyces cerevisiae were detected with the new mensuration method. The results indicates that the ratios ofβ-D-glucan in various strain Saccharomyces cerevisiae were distinctly difference （P≤0.05）. The most difference of cell wall and cell dry mass reached 138.10% and 189.52%, respectively.Theβ-D-glucans content of yeast must be influenced by culture medium and condition, so we optimized the Fermentation condition. Firstly, the influences of carbon source, nitrogen source, and enzyme activation on content ofβ-glucan were evaluated using factional factorial design. Based on this, the experiment for optimization of theβ-glucan fermentation medium was carried out with the design of the rotation-regression-orthogonal combination and received model equation: Y = 106.89+3.74X₁-8.85X₁²-5.72X₂²-7.96X₃²-7.52X₄². The optimal parameters were obtained by multinomial regression techniques as follows: glucose: 32.7 g/L, peptone: 18.9 g/L, yeast extract: 15.7 g/L;glycerin: 10.4 g/L. The optimized culture medium allowedβ-glucan to increase from 65.80 mg/100ml to 109.33 mg/100ml. Secondly, adopting orthogonal design to optimize theβ-glucan fermentation condition, the sequences of primary and secondary influence factor were quantity of culture medium, culture temperature, initial pH and quantity of inoculation. A satisfactory productivity of yeastβ-D-glucans was obtained under the condition: pH5.0, 5ml/100ml （quantity of inoculation）, 32℃and 60 ml/flask （quantity of culture medium）, the quantity ofβ-D-glucans increased from 109.33 mg/100ml to 128.30 mg/100ml.In the basis of optimize fermentation, the yeast biomass and quantity ofβ-D-glucans were simulated by Logistic, BoxLucas1 and SRichards2 model. The Logistic model mostly suitted to the yeast biomass, the simulation equation as follows: y = 1317. 3?1 +（1x2/0180..96）2.7, the correlation coefficient was 0.975. The best simulated model ofβ-D-glucans was SRichards2 model, the simulation equation as followed: the correlation coefficient was 0.996. The glucose consume model is founded with Exponemtial ExpDec1 model, which correlated experiment well with of R2 of 0.977, the simulation equationIn the past decades,β-D-glucans have been often isolated by an acid-alkaline method. However, using these methods had some drawbacks: the great mass ofβ-D-glucans were degraded and distributed in all levels in supernatant. This led to lowerβ-D-glucans yields compared with the original content in the cell walls and remarkably influenced on its biological function. A new mild method to extractβ-D-glucans from S.cerevisiae cells was proposed in this paper, which was composed of induced autolysis, water and organic solvent treatment, homogenization and protease hydrolysis. The broken yeast cell was key and difficulty in theβ-D-glucans isolation procedure, so yeast cells were firstly treated by hot water. Treated by hot water, the mechanical strength of the yeast cell wall decreased with the removal of the mannoproteins, which caused the yeast cell walls easy to be disrupted. As a result, the ratio of broken yeast cell slightly exceeded 95% after three passes in homogenization treatment in 70 MPa. Finally,β-D-glucans were obtained at a yield of 91% of the original ratio in the yeast cell walls and with a purity of up to 93% （w/w）. Because of infusibility,β-D-glucans was limited in the industry application.β-D-glucans modification was achieved by ultrasonication and homogeneous sulfation in this paper. The optical ultrasonical condition was 400W （power）, 24s （treat time）, 6s （intermission time） and 20 circle times. Using ultrasonication, the average diameter ofβ-D-glucans reduced from 56.49μm to 2.49μm.β-D-glucans sulfation reaction was in Urea-DMSO homogeneous system, in which 5% sulfate acid was employed as esterifiable reagent. The reaction carried through 4 h at 100℃, the sulfated yield was approximate 87.97% （w/w） and DS was 0.43 in the end.In order to make clear basic configuration ofβ-D-glucans and its sulfated derivative, some modern analysis technology FTIR, EA and NMR were adopted. These spectra revealed thatβ-D-glucans were （1→3）-linkedβ-D-glucopyranan backbone with （1→3）-β-branches every nine or five units and the etherification ofβ-D-glucans mainly exist on C6-SO3. Based on the elemental analysis, the empirical formula of sulfatedβ-D-glucans was （C6H10O5）20·9SO3·20H2O. The solution properties of sulfatedβ-D-glucans which was essential for the further insight on the correlation of structure to bioactivities, have not been reported. So based on YFY and KP wormlike cylinder models, the molecular parameters of sulfatedβ-D-glucans in solution were obtained by laser light scattering and viscometry in this paper. The results indicated that the conformational parameters of sulfatedβ-D-glucans were calculated to be 646 nm-1 for ML, 5.1 nm for q and 16.3 for C∞by wormlike cylinder mode, indicating a relatively extended flexible chain in the aqueous solution.Further, the immunobiological activity ofβ-D-glucans and its sulfated derivative was approved by mice experimentation. Preliminary immunopharmacological tests suggested that ultrasonic and sulfatedβ-D-glucans could significantly increased the ConA-induced spleen lymphocytes proliferation, the hemolytic level and the activity of NK cells （P≤0.01）.更多还原