【作者】 刘红芝；

【导师】 王强；

【作者基本信息】 中国农业科学院 ， 农产品质量与食物安全， 2009， 博士

【摘要】 酵母细胞壁占细胞干重的20-25%,甘露聚糖存在于酵母细胞壁外层,占细胞壁干重的40%左右,赋予细胞生物学活性和控制细胞壁孔径。甘露聚糖是免疫功能最强的酵母细胞壁多糖,它能增加动物体液免疫和细胞免疫能力,调节肠道菌群平衡,结合吸附外源性病原菌,并具有抗辐射、抗氧化、抗肿瘤等活性功能。本研究对不同酿酒酵母菌种的甘露聚糖含量进行了分析对比,结果表明酵母中甘露聚糖含量随其菌种不同而有明显差异(p<0.05)。原有从中科院微生物研究所购得菌株AKU 2.1424生物量最高,达905.02±9.91 mg/100ml;AS 2.0016甘露聚糖含量及其占细胞干重比率最高,分别为76.21±0.73 mg/100ml与12.92%。“实践8号”卫星搭载后的酿酒酵母样品,经过筛选得到的菌株AS 2.0016-M生物量增加46.69%,细胞壁占酵母干重比率增加3.81%,细胞壁厚度增加62.62%,甘露聚糖含量增加18.82%,β-葡聚糖含量增加146.87%,各指标均较原菌株增加达极显著水平(p<0.01)。对酿酒酵母AS 2.0016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:经二次多项式逐步回归分析确定了最佳培养基:蔗糖4.98 g/100ml、大豆蛋白胨4.39 g/100ml、酵母膏3.10 g/100ml、甘油2.21 g/100ml。使用优化后培养基得到的甘露聚糖由优化前的85.72±3.38 mg/100ml增加到162.53±3.47 mg/100ml,提高了96.48%。应用正交优化方法对培养条件进行优化,得出各因素对酵母甘露聚糖量的影响因素依次为:装液量>温度>起始pH>接种量。酵母最适的培养条件为:pH值5、接种量5 ml、温度32℃、装液量40 ml。对最佳培养条件进行验证试验,酵母甘露聚糖产量达到268.30±1.94 mg/100ml,较发酵条件优化前的162.53±3.47 mg/100ml,提高了65.08%。对热水抽提与碱法制备酿酒酵母甘露聚糖的工艺进行了优化,确定水提法制备甘露聚糖的最佳条件为:浓度25%(w/w)的酵母菌体在pH值6.5,3% NaCl和50℃的条件下振荡自溶24 h;然后将自溶后的菌体洗净,120℃处理3 h。碱提法制备酵母甘露聚糖的最佳条件为:KOH浓度2%、温度100℃、反应时间2 h。使用TCA去除酵母甘露聚糖中蛋白,蛋白脱除率可达70.94%。去蛋白后经两次醇沉,第一次乙醇浓度50%,复溶离心后使用体积分数为70%的乙醇进行二次醇沉。通过工艺条件优化,WSMP与ASMP粗品得率分别为6.42%与5.93%,纯度分别为81.32%与88.24%。通过凝胶过滤色谱纯化WSMP与ASMP,产品最终纯度达92.61%与93.31%。用HPLC测定WSMP与ASMP的平均分子量分别为1.81127×10~5 Da与6.5098×10~4 Da。采用GPC-LLS测定酵母甘露聚糖WSMP和ASMP的绝对分子质量,得到WSMP的重均分子质量(Mw)为1.667×10~5 Da,ASMP的重均分子质量为6.620×10~4 Da。WSMP的多分散系数为1.350,ASMP的多分散系数为1.17,二者均在1.5以下,说明两个样品的分子质量分布较为集中。单糖组成测定表明,酵母甘露聚糖主要由甘露糖组成,氨基酸分析表明,酵母甘露聚糖WSMP含有17种常见氨基酸,其中能够构成O-糖肽键的苏氨酸和丝氨酸有较高的比例,分别占总量的20.08%和11.46%,天冬氨酸(Asp)含量较高,占氨基酸总量的14.96%。通过β-消除反应、紫外光谱分析、氨基酸组成分析、气相色谱分析等手段,得出酵母甘露聚糖有N-糖肽键与O-糖肽键两种连接方式,且以N-糖肽键为主要连接方式。通过高碘酸氧化与Smith降解分析,采用FTIR和NMR测定,得出酵母甘露聚糖以α-(1,6)为主链,此外还含有大量α-(1,2)糖苷键。推测酵母甘露聚糖糖链结构为:通过原子力显微镜观察酵母甘露聚糖的立体形貌特征,看出WSMP是由许多小的圆形颗粒形成紧密的网络状结构,存在不同程度的聚集,ASMP聚集成近棒状。扫描电镜观察看出,甘露聚表面呈特征性胶联、聚集态的无定形结构。通过动物免疫增强试验,检测了WSMP、WSM和ASMP对脾淋巴细胞增殖反应、绵羊红细胞诱导小鼠迟发型变态反应(DTH)、小鼠腹腔巨噬细胞吞噬鸡红细胞等的影响。结果显示:WSMP高、中、低剂量组,WSM中、低剂量以及ASMP中剂量组可显著促进脾淋巴细胞增殖(p<0.05)。WSMP、WSM和ASMP均能增强绵羊红细胞诱导的小鼠迟发型变态反应,即可增强细胞免疫功能,其中以WSM的促进作用最为明显。低剂量的WSMP以及中、低剂量的WSM能显著增强小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬功能(p<0.05)。通过体外抗肿瘤实验,得出WSMP在1.666 mg/ml时能够抑制50%的HL-60肿瘤细胞,WSM也具有直接抑制HL-60生长的作用,但其活性略低于WSMP。体外抑制Eca-109与HepG2细胞生长实验结果显示,WSMP与WSM对这两种肿瘤细胞均有一定的抑制效果,WSMP作用效果优于WSM。ASMP体外无抗肿瘤作用。更多还原

【Abstract】 Yeast is surrounded by a tough, rigid cell wall that represents 20-25% of the dry weight of the cell. Mannan is one of the major components (together with glucan, chitin and protein) of the yeast cell wall, occupying about 35-45% of it deposited in the outside of the cell wall. The present of mannan not only provide the cells with rigidity that protects them from osmotic pressure but also helps to maintain their shapes during the cell cycle stage. It has been reported that most of the immunological effects observed within the intact yeast cells are reproduced with cell wall components and have been shown to be potent inducers of cellular and humoral immunity. Among them, mannan and mannoprotein are found to be the ones with significant activities. Besides, they could also balance the enterobacteria, combine with the extrinsic pathogen, resist tumour, acted as an anti-oxidant agent.The mannan contents in different S. cerevisiae strains were compared in this study, results showed that the mannan content to each strain was different. The biomass of AKU 2.1424 was 905.02±9.91 mg/100ml, it was higher than other strains, and the mannan conent of AS 2.0016 was the highest. Freeze-dried samples of four S. cescerevisiae strains were subjected to spaceflight. After the satellite’s landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (p<0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (p <0.05). The spaceflight mutant of AS 2.0016 showed 46.69%, 62.62%, 18.82% and 146.87% increment in biomass, cell wall thickness, mannan andβ-glucan content, respectively, when compared to the ground strain.The mannan content of yeast must be influenced by culture medium and condition, so we optimized the fermentation condition. On the basis of the single factor experiment, the carbon and nitrogen sources and enzyme activator were determined, and then the mathematical model was established by the quadratic rotary combination design, through response surface analysis. The regression equation obtained for the mannan production is as follows:The optimized concentrations of culture medium were determined as 4.98 g/100 ml sucrose, 4.39 g/100 ml soybean peptone, 3.10 g/100 ml yeast extract and 2.21 g/100 ml glycerol. The optimized culture medium allowed mannan production to be increased from 85.72±3.38 mg/100 ml to 162.53±3.47 mg/100 ml. The influence of culture condition on mannan production was evaluated and confirmed by orthogonale experimental design, the optimized culture condition was: pH-5, inoculum size-5 ml, temperature-32oC and media volume-40 ml. The maximum mannan production enhanced 65.08% at the optimum culture condition.The processing conditions of hot water extraction and alkali extraction of mannan were optimized. The optimum condition for hot water extraction was composed of induced autolysis at pH 5.0, 3% NaCl, 55oC, 120 r/min and hot water treatment.at 120oC for 3 h. Besides that, the The optimum condition for alkali extraction was determined as: KOH 2%, 100 oC and treated for 2 h. TCA was selected to remove the freeprotein, after that, ethanol was used to precipitate the mannan. Finally, WSMP and ASMP were obtained at a yield of 6.42% and 5.93%, purity of 81.32% and 88.24% respectively.The physicochemical properties of WSMP an ASMP were studied. The average Mw of WSMP and ASMP were 1.81127×105 Da and 6.5098×104 Da, the weight-average molecular of WSMP and ASMP were 1.667×105 Da and 6.620×104 Da respectively. Both the polydispersity to each mannan were less than 1.5, that meaned the molecular mass with narrow distribution. HPLC analysis showed that mannan mainly consist of mannose. WSMP included 17 amino acid and the ratio of Ser, Thr and Asp were higher.β-elimination reaction, UV, amino acid analysis and GC analysis showed that the mannoprotein linked to oligosaccharides by O-linked and N-link oligosaccharides. The structure of mannan was analysed by FTIR and NMR specturm and determined as follows:By AFM, it was observed the netted texture of WSMP consisted by little round particles and existed as a aggregation, ASMP aggregated to rod-shaped. By SEM, the mannan appearance also showed glue-link and aggregation state.Effects of WSMP, WSM and ASMP on splenic lymphocyte transformation assay, delayed type hypersensitivity reaction and peritoneal macrophage phagocytizing chicken red blood cell assay were investigated in mice by immune-enhancing assay of animals. Results indicated that the WSMP, WSM and ASMP affected on on splenic lymphocyte transformation assay significantly (p<0.05). Delayed type hypersensitivity reaction was significantly enhanced when the mice were administered with WSMP, WSM and ASMP. Therefore, both WSMP, WSM and ASMP have immunization regulating function which could be judged, and overall evaluation was that the immune function of WSM and WSMP was stronger than that of ASMP. The WSMP and WSM had different degrees of inhibitory effects on HL-60, Eca-109 and HepG2 tumor cells in vitro.更多还原