【作者】 刘媛媛；

【导师】 周志江；

【作者基本信息】 天津大学 ， 食品科学， 2012， 硕士

【摘要】 赤藓糖醇是分子量最小,热量值最低(≤1.66kJ/g)的糖醇,具有优良的物理化学性质和保健功能。赤藓糖醇的生产方法有化学合成法、微生物发酵法和生物提取法等,其中发酵法是目前工业化生产赤藓糖醇的唯一办法,而如何选育高产的发酵菌株及确定最优化的发酵条件是工业化生产的关键。本研究结合了微生物的化学诱变方法和物理诱变方法,分别使用化学诱变剂硫酸二乙酯(DES)和紫外线,对赤藓糖醇产生菌丛梗孢酵母进行诱变,并通过薄层层析法、分光光度法和高效液相色谱法对发酵产物进行定性和定量分析,筛选出赤藓糖醇高产菌株并验证其遗传稳定性。通过单因素试验和正交试验方法确定高产诱变菌株的最适发酵条件。发酵液定性试验结果表明,发酵产物主要为赤藓糖醇,此外还含有极少量甘油和核糖醇。通过试验建立了初步测定赤藓糖醇和葡萄糖含量的分光光度法,即高碘酸氧化法和DNS法,以及精确定量的高效液相色谱法。通过DES化学诱变筛选出菌株D49,赤藓糖醇产量为69.58g/L,较野生菌提高19.2%,葡萄糖-赤藓糖醇转化率为28.1%。对菌株D49进行紫外诱变,筛选得到高产诱变菌株D49-UV53,赤藓糖醇产量为86.25g/L,较D49提高了23.87%,较野生菌提高了47.8%,葡萄糖-赤藓糖醇转化率达31.44%。突变菌株D49-UV53经连续8次传代后,测定每代的赤藓糖醇产量,其偏差范围为±2.5%,证明该菌株有很好的遗传稳定性。在单因素试验的基础上设计正交试验,对高产诱变菌株D49-UV53进行发酵工艺优化。优化后的赤藓糖醇产量可达116.35g/L,是优化前(86.25g/L)的1.3倍,野生菌产量(58.36g/L)的2.0倍,葡萄糖-赤藓糖醇转化率为34.10%。赤藓糖醇作为一种新兴功能性甜味剂,在食品、化妆品、医药、化工等领域具有广阔的市场前景。本研究筛选所得诱变菌的赤藓糖醇产量较出发野生菌种具有较大幅度的提高,有望用于赤藓糖醇的工业化生产。更多还原

【Abstract】 Erythritol is a kind of polyols with the smallest molectular weight and lowest calorie. It has good physical and chemical characteristics. Erythritol can be produced by chemical combination, microbial fermentation and biological extraction. And microbial fermentation is the only way that is being used in erythritol’s industrial production. So it’s important to find out effective ways to select high-yeild strains for the industrial production, and to optimize their fermenting conditions.This research combined the chemical and physical mutating methods, using both ultraviolet (UV) rays and the mutagens of diethyl sulfate (DES) to cause mutagenicity. Fermentation products were measured qualitively and quantitatively through different methods, such as thin layer chromatography (TLC), spectrophotometry and high performance liquid chromatography (HPLC). After checking out the hereditary stability of the high-yeild mutagenesis strain, single factor experiment and orthogonal test were done to find out the best fermenating conditions for the strain.The qualitative tests showed that the main component of the fermentation product was erythoritol, with low amount of by-products of glycerol and ribitol. Periodic acid and 3, 5-dinitrosalicylic acid (DNS) colorimetry were established to make preliminary measurement of erythritol and glucose, while HPLC was established to make accurate detection.Strain named D49 was selected through chemical mutagenesis with DES. Its erythritol production was 69.58g/L, which was 19.2% higher than the wild type strain. And the percent conversion from glucose to erythritol was 28.1%. Strain D49 then went through the UV mutagenesisi and a strain named D49-UV53 was selected. Its erythritol production was 86.25g/L, which was 23.87% higher than the strain D49, and 47.8% higher than the wild type strain. Strain D49-UV53’s percent conversion from glucose to erythritol reached 31.44%. The yield of erythritol was stable with a relative deviation of±2.5% during the continuous cell culture for 8 genetations, which showed good hereditary stability.Single factor design and orthogonal design were used to optimize the strian’s culturing conditions. After fermentating condition optimization, erythritol production of strain D49-UV53 could reach 116.35g/L, which was 1.3 times of the production before optimizing (86.25g/L)and 2.0 times of the wild type strain(58.36g/L).As a new functional sweetener in the world, it has many potential uses in areas, such as food, cosmetic, pharmaceuticals and chemical industries. High-yield strain was found and the production of erythritol had been improved greatly through this research. And it may be used in the industrial production after futher studies.更多还原