论文总字数：20620字

摘 要

甘露醇是一种天然的六碳糖醇，由于其具有热量低，甜度适宜，无毒副作用等特性，是重要的食品添加剂。D-乳酸是一种天然的手性分子，在新材料、制药、农药等领域具有广泛的应用，具有较高的附加值。在厌氧条件下假肠膜明串珠菌（Leuconostoc pseudomesenteroides）进行的是异型乳酸发酵，可产生甘露醇和D-乳酸两种产物，这大大提高生物法制备甘露醇的经济性。果葡糖浆是由淀粉水解和异构化制成，其中同时含有果糖与葡萄糖，是一种常用的甜味剂，而且价格相对低廉，因此以果葡糖浆为原料发酵制备甘露醇和D-乳酸具有可行性。

本文以假肠膜明串珠菌Leuconostoc pseudomesenteroides G123作为生产菌株，将果葡糖浆作为碳源制备甘露醇与D-乳酸。考察了不同果葡糖浆制备种子的性能，随后研究了柠檬酸盐、表面活性剂和发酵温度对菌株发酵性能的影响，并比较了不同pH调控方式对生产性能的影响。

相关研究显示：55%的果葡糖浆可作为种子培养基的碳源，当柠檬酸铵的添加量为2g/L，甘露醇产量可增加31.4%；而添加表面活性剂吐温80并不能显著提高甘露醇产量。发酵试验表明：较高的温度有利于指数期菌体代谢性能的提高，但在静止期，细胞代谢活力衰竭更显著，最终甘露醇、D-乳酸的产量分别为61.07g/L和18.03 g/L，产物对底物的总得率仅为80.7%。随后将前期pH控制在pH5.5，后期维持pH4.5，指数期的菌体代谢性能进一步提高，底物消耗速率提高了40%，甘露醇生产速率达到1.92 g/(L.h)，提高了31.5%，最终甘露醇对果糖的得率为89.8%，甘露醇与D-乳酸的产量分别达到57.7g/L和27.1g/L。

在此基础上对发酵液中甘露醇的提取开展了初步研究，采用微滤、浓缩、预脱色和粗结晶的方式可将大部分杂质除去，粗晶体经脱色与重结晶精制后可获得甘露醇产品，纯度达99.8%，不含D-乳酸，产品的熔点为165～167℃，符合相关质量标准。整个过程中的甘露醇总提取收率为75.5%，产品损失主要集中于粗结晶和脱色处理过程。本论文的相关成果可为相关工艺的改进与完善提供借鉴。

关键词：果葡糖浆，甘露醇，D-乳酸，发酵，提取

Mannitol and D-lactic acid production from high fructose corn syrup by anaerobic fermentation

ABSTRACT

Mannitol is a natural six-carbon sugar alcohol. It is an important food additive due to its low heat, sweetness appropriate, without caries, non-toxic side effects and other characteristics.D- lactic acid is a natural chiral molecules, which has been used in the field of new materials, pharmaceuticals, pesticides, etc, so it has high added value. Leuconostoc pseudomesenteroides is heterolactic fermentative bacteria under anaerobic conditions, which can produce two products mannitol and D- lactic acid and it is benefited to improve the economy of biomannitol. High fructose corn syrup is made from starch hydrolysis and isomerization, which contains both fructose and glucose. It is a kind of common sweeteners and its price is relatively low. Therefore, it is feasible to produce mannitol and D- lactic acid by fermentation with high fructose corn syrup as raw material.

The strain Leuconostoc pseudomesenteroides G123 was used in this work, and High fructose corn syrup was prepared as a carbon source for mannitol and D- lactic acid production. We compared the performance of different high fructose corn syrup in seed cultivation, then the effects of the citric acid salt, surface active agent on the fermentation were studied, the fermentation temperature and pH control strategy were also discussed.

Related research showed 55% of high fructose corn syrup could be used as seed medium carbon source. When the addition amount of ammonium citrate was 2 g/L, mannitol yield increase 31.4%; while adding a surfactant Tween 80 did not increase mannitol yield significantly. Fermentation tests showed higher temperatures improved the performance of bacterial metabolic, but cell viability declined more significantly in resting, the final mannitol and D- lactic acid were 61.07 g/L and 18.03 g/L. The total yield of the substrate was 80.7%. Then pH was controlled in pH5.5 in exponential phase, and maintained pH4.5 in stationary phase. Metabolic performance in exponential phase was enhanced, the substrate consumption rate was increased by 40%. Mannitol productivity was increased to 1.92 g/(Lh), which improved 31.5 % . The final yield of fructose to mannitol was 89.8%. Mannitol and D- lactic acid reached 57.7 g/L and 27.1 g/L.

The extraction of mannitol from fermentation broth was also studied, microfiltration, concentration pre-discoloration and coarse crystallization could remove most of the impurities, the crude crystals were decolorized and recrystallized .Mannitol purity was 99.8%, and without D- lactic acid. The melting point of the product was 165~167 ℃, meeted the relevant quality standards. The extraction yield of mannitol was 75.5% in the entire extraction process. Main loss of product was happened in coarse crystallization and decolorization process. These results can provide a reference for improvement of production technology.

Keywords: Fructose fructose corn syrup; Mannitol; D- lactic acid; Fermentation; Extraction

目录

摘 要 I

Abstract II

第一章 文献综述 1

1.1 甘露醇的结构性质、应用和传统生产方法 1

1.2 D-乳酸的结构性质、应用和传统生产方法 2

1.3 假肠膜明串珠菌与异型乳酸菌发酵 2

1.4 明串珠菌制备甘露醇的研究进展 3

1.5 本文的研究目的与内容 4

1.5.1 研究目的 4

1.5.2 研究内容 5

第二章 实验材料与方法 6

2.1 实验材料 6

2.1.1 菌株 6

2.1.2 实验试剂 6

2.1.3 实验仪器 7

2.1.4培养基 8

2.2 实验方法 9

2.2.1 种子培养 9

2.2.2 摇瓶发酵 9

2.2.3 上罐发酵 9

2.2.4 果葡糖浆替代果糖培养种子的研究 9

2.2.5 柠檬酸铵对甘露醇发酵影响的研究 9

2.2.6 吐温80对甘露醇发酵性能影响的研究 10

2.2.7 不同温度下甘露醇发酵性能的研究 10

2.2.8 分阶段pH调控甘露醇发酵性能的研究 10

2.3 甘露醇的分离提取 10

2.4 分析与检测方法 11

2.4.1 菌体密度的测定 11

2.4.2 糖含量与产物的测定 11

2.4.3 色素含量的测定 11

第三章 结果与讨论 12

3.1 果葡糖浆对种子培养的影响 12

3.2 柠檬酸铵对发酵性能的影响 12

3.3 吐温80对发酵性能的影响 13

3.4 温度对发酵性能的影响 13

3.5 分阶段pH调控对发酵性能的影响 15

3.6 发酵液中提取甘露醇 17

第四章 结论与展望 19

4.1 结论 19

4.2 展望 19

参考文献 20

致谢 23

第一章 文献综述

1.1 甘露醇的结构性质、应用和传统生产方法

甘露醇是一种六碳糖醇，广泛存在于藻类和各类植物中，又名甘露糖醇、或木蜜醇。分子式是C6H₁₄O₆，分子量为182.17。易溶于水，是一种白色透明的固体，有甜味^[1-4]。溶解度为18 g/100 g（25℃），微溶于乙醇，较多地溶于热水，溶于吡啶和苯胺，不溶于醚。水溶液呈酸性。其化学性质比较稳定，不易被空气氧化，对稀酸和稀碱稳定。

图1-1 甘露醇结构式

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