【作者】 唐锐；

【导师】 吴振强；

【作者基本信息】 华南理工大学 ， 发酵工程， 2017， 硕士

【摘要】 红曲色素是聚酮类的天然化合物,具有良好的酸碱稳定性、热稳定性和耐光性。它能够减少糖尿病和肥胖病发病率,具有较好的应用前景。红曲色素包括红曲黄色素、红曲橙色素、红曲红色素。萃取发酵是一种高效生产红曲色素的新型技术。红曲霉菌Monascus anka GIM 3.592萃取发酵主要产生的是黄色素和橙色素。在萃取发酵中,表面活性剂Triton X-100改变细胞膜渗透性,增加胞内红曲色素渗透至胞外的速率,将胞内红曲色素萃取至胞外,胞内色素产物浓度降低,能有效解除胞内色素反馈抑制现象,进而极大地提高了红曲色素的产量。但色素存在于表面活性剂Triton X-100的胶束结构中,对色素的后续分离和纯化造成困难。针对萃取发酵液中红曲色素与表面活性剂Triton X-100难以分离纯化的问题,本研究尝试了几种不同的方法来分离红曲色素与表面活性剂Triton X-100。(1)色素衍生法:通过在萃取发酵液中添加谷氨酸钠,将弱极性的橙色素衍生化为极性的红色素。再依次通过有机溶液萃取、水溶液反相萃取,实现了红曲红色素在水相中的富集,红曲红色素的回收率达到73.4%。同时,表面活性剂在二氯甲烷相中富集,回收率为96%。使用衍生法不仅可以提高红色素的含量和色素产品品质,还增加了萃取方法分离红曲色素的效率和选择性。(2)改性填料层析法:通过常规硅胶填料与脂肪酸酯类的纳米纤维素共混,可以增加或者减少柱层析填料的亲水性能。乙酸乙酯和甲醇溶液洗脱负载于混合填料中的色素和表面活性剂时,红曲色素和表面活性剂依次被洗脱和分离。对填料进行低成本的物理改性,使得柱层析方法对色素和表面活性剂分离的选择性得到增强,一定程度上改善了红曲色素与表面活性剂的分离效果。(3)有机溶剂萃取-碱性大孔树脂吸附法:三氯甲烷萃取色素发酵液后,再通过碱处理的极性大孔树脂S-8选择性地吸附溶剂中游离的红曲色素。碱性S-8对最大吸收峰为470 nm的色素的吸附率达到95.06%,而对表面活性剂的吸附率仅为2.53%。吸附在树脂中的色素可以被盐酸的甲醇溶液洗脱,总洗脱率可达80%左右。回收的S-8可被再生并重复使用,吸附效率无明显下降。研究表明,在三氯甲烷溶液中,树脂对色素吸附的过程符合Freundlich热力学吸附平衡模型和准二级动力学模型。根据Weber and Morris粒内扩散模型推测,色素的吸附的速率受到膜扩散和颗粒内扩散等步骤的限制。该方法为有效分离和纯化萃取发酵液中的色素提供了新途径。更多还原

【Abstract】 Monascus pigments are natural compounds containing polyketones structures.They have excellent thermal stability as well as resistance to photodegradation and pH change.Representing a good application prospects,they can also reduce the incidence of diabetes and obesity.Monascus pigments are mainly composed of yellow pigments,orange pigments and red pigments.Extractive fermentation was an advanced technology for a high-yield productivity of Monascus pigments.Yellow pigments,orange pigments are mainly produced by Monascus anka GIM 3.592 in extractive fermentation.Surfactant Triton X-100 changed the cell membrane permeability and then increased the penetration rate from intracellular pigments to extracellular.Intracellular pigments could be extracted to the extracellular broth,and the decrease of intracellular pigments could relieve feedback suppression phenomenon effectively,thus high extracellular Monascus pigments concentration were achieved.However,the solid bonding between pigments and Triton X-100 micelles challenged a subsequent separation and purification of the pigments from fermentation broth.Several different methods have been proposed to separate the Monascus pigments from the surfactant Triton X-100 aimed at the difficulty of separation of Monascus pigments and surfactant in extractive fermentation broth.(1)Derivatives of pigments:The weakly polar orange pigments were derivatized to polar red pigments after adding sodium glutamate in extractive fermentation broth.Then by means of organic solution extraction and aqueous solution back-extraction,the red pigments was enriched in water phase with a recovery of 73.4%,and the surfactant was enriched in the dichloromethane phase with a recovery of 96% at the same time.The use of derivatization can not only improve the content and quality of red pigments,but also increase the efficiency and selectivity of the separation of Monascus red pigments.(2)Physical modified chromatography method: By blending the conventional silica gel filler with fatty acid ester nano-cellulose,it is possible to increase or decrease the hydrophilicity of the column chromatography.When ethyl acetate and methanol solution were used to elute the pigments and surfactant loaded on the mixed filler,Monascus pigments and surfactant are eluted and separated in turn.The low-cost physical modification of the filler enhanced the selectivity of the separation of the pigments and the surfactant by the column chromatography,and improved the separation effect of the Monascus pigments and the surfactant to a certain extent.(3)Organic solvents extraction and subsequent adsorption using S-8 alkali resin: The extractive fermentation broth was extracted by chloroform and subsequent using S-8 alkali resin selectively adsorbed free Monascus pigments in the solvent.The adsorption rate of S-8 alkali resin to Monascus pigments with the maximum absorption peak of 470 nm was 95.06%,while the adsorption rate of surfactant was only 2.53%.respectively.The use of methanol solution containing HCl led to the elution efficiency with a total pigment recovery yield of 80%.Moreover,the recovered S-8 can be regenerated and reused without significant decrease in adsorption efficiency.The pigment adsorption could be described by Freundlich equilibrium model with a pseudo-second order kinetic course in chloroform solution.According to the Weber and Morris diffusion model,it is speculated that the rate of the pigment adsorption is limited by steps such as membrane diffusion and intra-particle diffusion.The method was demonstrated to be a new approach for pigment isolation in extractive fermentation.更多还原