【作者】 贾伟；

【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(erythritol)是一种新型食品甜味剂。具有甜味纯正,热量值极低,稳定性高,生物耐受性高及抗龋齿等优点。可作为低能量保健品配料应用于食品生产,并且在医药及化工领域得到应用,表现出良好的应用前景。本实验室前期从蜂蜜样品中分离得到一株丛梗孢酵母(Moniliella sp.),发现具有产赤藓糖醇的能力。本研究的目的在于:考察菌株发酵培养基及发酵条件对赤藓糖醇产量的影响,从而对其产赤藓糖醇的条件进行优化,建立从发酵液中分离提取赤藓糖醇的方法。在丛梗孢酵母赤藓糖醇产生条件优化实验中,首先测定菌体生长曲线,获得最优种子培养基为葡萄糖20 %,酵母膏1 %,在此条件下将培养48 h的种子液用于发酵培养。通过单因素实验,碳源、氮源、无机盐、高浓度NaCl、初始pH值、接种量、温度、发酵时间等因素对赤藓糖醇产量的影响。发现丛梗孢酵母的最适碳源为葡萄糖,浓度为35 %;最适氮源为酵母膏,浓度为1 %;添加磷酸二氢钾、硫酸镁、氯化钙等无机盐可提高赤藓糖醇产量。最适初始pH为5.0,接种量5 %,温度为30℃,发酵9 d。根据单因素试验的结果,设计正交试验,用SPSS软件进行结果分析,表明最优发酵培养基组成为:葡萄糖30 %、酵母膏1 %、CaCl20.2 %,最佳发酵条件为:接种量1 %,发酵培养基初始pH 6.0,30℃摇瓶培养9 d。在此条件下进行发酵试验,赤藓糖醇的产量可达110.61 g/L。在赤藓糖醇分离提纯的实验中,首先通过钝化灭酶、离心去除发酵液中的菌体及大分子蛋白质。其次,采用活性炭脱色,脱色率达97.38 %。经超滤截除蛋白质等大分子物质后,采用阴阳离子交换树脂处理去除发酵液中残留的金属盐离子、色素及多元醇等,试验表明最佳层析条件为:上样量5 mL,洗脱速度1.0 mL/min,收集20~65 min的流出液。对流出液进行组分分析,赤藓糖醇占干物质含量为92.48 %。流出液进一步用旋转蒸发器进行蒸发浓缩,至赤藓糖醇含量为60 g/100 mL以上时,令其缓慢冷却。向浓缩液中加入0.05 %晶核,在10℃下放至24 h,出现结晶。回收母液,重复结晶两次,将晶体过滤、洗涤后干燥、称重,赤藓糖醇的回收率为74.35 %。采用高效液相色谱法检测产品成分,赤藓糖醇纯度为97.62 %。本研究通过单因素和正交试验,阐明了丛梗孢酵母赤藓糖醇产生的最优条件,发现该菌株容易培养,赤藓糖醇产量较高。并初步建立了从发酵液中分离提取赤藓糖醇的方法。为利用丛梗孢酵母进行赤藓糖醇的工业化生产提供了依据。更多还原

【Abstract】 Erythritol is a new kind of sweetener which has many advantages such as pure sweet taste, extremely low heat, high stability, highly tolerated by the intestinal, and prevention to dental caries. It has a broad prospect in the food industry as low energy food additive, and can be also well used in medical industry and chemical industry. An erythritol producing microorganism strain from a bee honey sample has been identified as Moniliella sp. in a previous study in our laboratory. One purpose of this study is to optimize the fermentation medium and conditions for the strain; the other purpose is to establish the separating and purifying method of erythritol from the fermentation broth.In the optimizing experiments, a 48 hours culture was determined as inolulum seeds according to the growth curve of Moniliella sp. BH010. Then fermentation factors such as carbon sources, nitrogen source, inorganic salt, NaCl concentration, initial pH value, inoculums, temperature, and fermentation time were studied by single-factor experiment. The results indicated that the best carbon source of Moniliella sp was glucose with the optimal concentration of 35 %, and the best nitrogen source was yeast extract with the optimal concentration of 1%. The addition of some mineral salt to the media such as KH2PO4, MgSO4 and CaCl2 increased the production of erythritol. The optimal initial pH was found to be 6.0, inoculum amount 5 %, fermentation temperature 30℃, and cultivate time 9 days. Then the orthogonal experiments of medium component and fermentation condition were further applied to find the optimal fermentation conditions. According to the result analyzed by SPSS , the optimal fermentation medium consisted of 30 % glucose, 1 % yeast extract, 0.2 % CaCl2. The optimal fermentation conditions were: a 48 h culture as inoculation seeds, inoculum amount 1 %, initial pH 6.0, culture temperature 30℃, and culture time 9 days. The yield of erythritol could reach 110.61 g/L under these conditions.In the experiment of separation and purification of erythritol, cells and mycelium in the broth were removed by deactivating enzyme and centrifuged. Discoloration of the broth was carried out by using activated carbon powder and the discoloring rate could reach 97.38 %.After the entraps of the large proteins and left minute carbon power by ultra filtration membrane, ions-exchange resins was used to remove the metal mineral salt, pigment, organic substances and so on. The quantity of sample was 5 mL, eluting speed 1.0 mL/min, and the collecting time 20 to 65 minues. According to the component analysis of effluent, 92.48 % of dry substance was erythritol, and the impurities were almost removed. The effluent then was concentrated by rotary evaporation to the erythritol concentration more than 60 g/100 mL. 0.05 % crystal nucleus were added while the concentrate was cooling. The concentrate stood at 10℃for 24 hours, and then the crystals formed. Crystal and bottom liquid were separated by centrifugalization, the bottom liquid was recycled and re-crystallized twice and crystals were collected and dried at 45℃until the quantity was stable. The recycle rate was calculate by quantifying the quantity of the total erythritol crystal, and the result was 74.35 % .The purity of erythritol crystal was 97.62 % evaluated by the HPLC measurement result.With single-factor experiments and orthogonal experiments, the study illustrated the optimal fermentation condition for a erythritol-producing Moniliella strain. It was found that Moniliella sp. BH010 has a high production of erythritol under simple conditions. A primary method to purify erythritol from the fermentation broth was also established. This research provided theoretical basis to the industrial production of erythritol.更多还原